gene transfer - bio-rad.com · gene pulser® electroprotocol 086 survey number cell type...

gene transferElectroprotocols Species List

Mammalian Cells Survey Number(s)

B-cell line, unspecified .......................................................... 121

Hamster, CHO, ovary ........................................ 85–91, 156, 168

Human, 293, kidney ..................................................... 108, 156

Human, B lymphomas: BJAB, P3HR-1, B95-8 .................... 157

Human, C-4I, cervical carcinoma ......................................... 155

Human, CEMx174, T lymphoblastoid ...................................... 92

Human epithelial cells ........................................................... 158

Human, fibroblast ......................................................... 160, 163

Human, GCT, fibrous histocytoma .......................................... 93

Human, HEL cells, eythroleukemia ....................... 158, 166, 192

Human, HeLa, epithelial carcinoma ...........................94–98, 158, 167, 192, 196

Human, Hep3b2, hepatocytes ....................................... 98, 163

Human, HepG2, hepatoma ............................................ 99, 161

Human, HL 60, eythroleukemia ............................................ 166

Human, HuT78, cutaneous T-cell lymphoma ....................... 100

Human, JEG-3, choriocarcinoma ......................................... 101

Human, Jurkat ...................................................................... 107

Human, JY, B-cell, Epstein-Barr virus transformed .............. 102

Human, K562, chronic myeloid leukemia ......................103–106, 166, 168, 192

Human, lymphoblast cell lines, EBV immortilized ................. 108

Human, lymphocytes, primary .............................................. 194

Human, MCF-7, breast...........................................................110

Human, MRC-5, lung ......................................................111, 112

Human, pancreatic ................................................................113

Human, Raji, Burkitt lymphoma ............................................ 107

Human, red blood cells ..........................................................114

Human, squamous cell carcinoma, oral & cervical lines ............................................................... 162

Human, T-cells ...............................................................107, 159

Human, TCCSUP (epithelial-like) bladder carcinoma ............ 153


Human, U373, glioblastoma ..................................................115

Human, U937, hystiocytic lymphoma .................... 106, 116, 117

Human, UC729-6, lymphoblastoid, B-cells ...........................118

Human, V79, skin cells, fibroblasts ........................................119

Hybrid, rat/mouse, MEL cells................................................ 168

Hybrid, mouse/human , A9 fibroblast ................................... 165

Hybridomas .......................................................................... 164

Monkey, COS, kidney ............................ 126–128, 154, 161, 195

Monkey, CV-1, kidney ........................................................... 167

Monkey, Vero, kidney .............................144, 155, 158, 160, 162

Mouse, 3T3, embryo .................................................... 160, 167

Mouse, 32d, myeloma .......................................................... 134

Mouse, A-9, derivative of mouse L cell ................................. 120

Mouse, BALB/c 3T3, clone A31, fibroblast, embryo ............ 122

Mouse, BbSutA, hematopoietic............................................ 166

Mouse, C127, fibroblast, mammary tumor .....................125, 193

Mouse, C2, muscle myoblast ............................................... 123

Mouse, C2C12, muscle .........................................................124

Mouse, D10.G4.1, T-cell, helper ............................................ 143

Mouse, embryonic stem cells ....................................... 129, 130

Mouse, erythroleukemia cells ........................................131, 132

Mouse, FDC-PI, Il-3-dependent cell line ............................... 133

Mouse, J558-L, myeloma ............................................. 134, 135

Mouse, L-cells ....................................................................... 163

Mouse, L929, connective tissue ........................................... 109

Mouse, LM(TK-), connective tissue ................................136, 137

Mouse, mammary epithelial cells .......................................... 154

Mouse, NIH/3T3; embryo ............................................. 139, 159

Mouse, NSO, myeloma cells ................................................. 140

Mouse, p3x63AG8; myeloma ............................................... 167

Mouse, SP2/0, [Sp-2], myeloma .................................... 141, 142

D035551


Mouse cells, unspecified ...................................................... 146

Mouse, WEHI-3B, myelomonocytic leukemia ....................... 138

Mouse, X-63, myeloma [p3 X63 - Ag8.653] .......................... 145

Ovine (sheep), CSL503, fetal lung ......................................... 152

Ovine (sheep), R.E., rumen ....................................................151

Rat-1 ..................................................................................... 157

Rat brain ............................................................................... 195

Rat, CA77, medullary thyroid carcinoma cell line .................. 197

Rat, D202CC, hepatoma ...................................................... 153

Rat, fibroblasts ...................................................................... 148

Rat, H4-11-E-C3, hepatoma ................................................. 150

Rat, L-6, myoblast ................................................................. 149

Rat, N62 T cells ................................................................... 154

Rat, PC12, adrenal pheochromocytoma ................................ 95

Rat, submandibular acini (secretory cells) ..............................147

Gene Pulser® Electroprotocol

085

Survey Number

Mammalian, adherentCell Type

Hamster, CHO -ATS49, ovary SpeciesUsed

DNA: pAG-6 (pSV2-gpt derivativecontaining hamster APRT), 8.4 kB, linear.

MoleculesElectroporated

DMEM + 10% Fetal Bovine Serum + NonEssential Amino Acids (NEAA)+ Pen/Strep(GIBCO/BRL, Sigma)

Cell Growth Medium Log phaseGrowth Phase atHarvest

TD (analogous to Phosphate Buffered Saline)Wash Solution

10 min., 4°CPre-pulseIncubation

4 °CElectroporationTemperature

Berg buffer ( HEPES Buffered Saline- seenotes)

ElectroporationMedium

Not givenCell Density

10 (7) cells / 0.8 mlsVolume of Cells

1µg / µlDNA Concentration

TE (10 mM Tris, 1 mM EDTA,pH 8.0)

DNA ResuspensionBuffer

20 µlVolume of DNA

Gene Pulser® apparatus Instruments Used

0.4 cmCuvette Gap

0.8 kVVoltage

2.0 kV/cmField Strength

25 µFCapacitor

(Pulse Controller) Ω none Resistor

0 .9 msecTime Constant

DMEM + 10% Fetal Bovine Serum + NEAA +Pen/Strep

Outgrowth Medium

37 °COutgrowth Temperature

14 daysLength of Incubation

Alanosine, Azaserine, Adenine ( aprt+) or HAT (gpt+)

Selection Methodor Assay Used

2 x10 (7) cells / 20 µgElectroporationEfficiency

usually high %Per Cent Survival

Sandra PenningtonName of Submittor

Baylor College of MedicineBiochemistry1 Baylor PlazaHouston, TX 77030

InstitutionAddress

Note: exponential values designated in parentheses.

Pennington, S.L., and Wilson, J.H. (1991) Gene targeting inChinese Hamster Ovary cells is conservative. PNAS 88:9498-9502.

HBS: 10mM HEPES, pH 7.2,150 mM NaCl, 5 mM CaCl2

Relevant Publications and/or Comments

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After the Pulse

Gene Pulser® Electroprotocols

* We recommend adapting this protocol to use the Gene Pulser electroporation buffer (catalog #165-2676, 165-2677), which increases cell viability and transfection efficiency in mammalian cell lines.

Medium*


086

Survey Number

Mammalian,adherentCell Type

Hamster, CHO -K1, ovary, (requires proline)SpeciesUsed

DNA: 11 to12 kB expression vector,Rep 4.


F12 + 10% serum (GIBCO/BRL, Sigma)Cell Growth Medium Log phaseGrowth Phase atHarvest

Phosphate Buffered SalineWash Solution

10 min., on ice.Pre-pulseIncubation

Room temperature (pulse), then iceElectroporation

Temperature

Phosphate Buffered SalineElectroporationMedium

2 x 10 (5) cells / pulse Cell Density

0.8 mlVolume of Cells

0.5 µg / pulse DNA Concentration

Not givenDNA ResuspensionBuffer

Not givenVolume of DNA

Gene Pulser® apparatus & CapacitanceInstruments Used

0.4 cmCuvette Gap

0.20 to 0.40 kVVoltage

0.5 to 1.0 kV/cmField Strength

500 & 960 µFCapacitor


18 to 22 msecTime Constant

F12 + serum + hygromycin or neomycinOutgrowth Medium


14 to 21 daysLength of Incubation

Hygromycin or neomycin selectionSelection Methodor Assay Used

Not done as yet - just beginningElectroporationEfficiency

>10%Per Cent Survival

Not listedName of Submittor

InstitutionAddress

Note: exponential values designated in parentheses.Relevant Publications and/or Comments

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Before the Pulse

After the Pulse



Medium*


087

Survey Number


Hamster, CHO, ovary SpeciesUsed

DNA: supercoiled DNA used for transienttransfections; linearized DNA used forstable transfections.


F12, 10% Fetal Calf Serum (FCS)(GIBCO/BRL, Sigma)

Cell Growth Medium 50 to 70% confluencyGrowth Phase atHarvest

Wash two times in electroporation bufferWash Solution

4°C, 10 min. (option: add 50µl FCS ifusing HeBS as electroporationmedia; 50 µl salmon sperm DNA fortransient transfections).

Pre-pulseIncubation

Room temperatureElectroporation

Temperature


5 x 10 (6) cells/pulse for transient assay;Cell Density


10 µg / pulseDNA Concentration

Not given; final volume: 0.8 mlDNA ResuspensionBuffer

Not given; final volume: 0.8 mlVolume of DNA


0.4 cmCuvette Gap

1.30 kVVoltage


25 µFCapacitor

(Pulse Controller) Ω none. NOTResistor

0.4 msecTime Constant

F12, 10% Fetal Calf Serum (FCS) Outgrowth Medium


48 to 72 hrs.Length of Incubation

G418 (stable transfections) andtransient assays


Not givenElectroporationEfficiency

about 50%Per Cent Survival

Jackie BeallName of Submittor

University of AdelaideDepartment of BiochemistryP.O. Box 498Adelaide 5001Australia

InstitutionAddress

Note: exponential values designated in parentheses.**It is NOT RECOMMENDED to use high voltage with out thePulse Controller.Note: Stable transfections generally do not use carrier DNA.Also, the level of selective agent required to kill offnon-transfected cells needs to be established beforetransfection. The level required should kill non-transfected cellsin approximately 7 days.PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES, pH 7.2, 150 mM NaCl, 5 mM CaCl2


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Medium*


088

Survey Number

Mammalian, adherent, suspensionCell Type

Hamster, CHO, ovarySpeciesUsed

DNA: linearized plasmids, about10 kB.


Hams F12 (Gibco)Cell Growth Medium 70 % confluenceGrowth Phase atHarvest

Phosphate Buffered Saline, pH 7.4Wash Solution

10 min. on icePre-pulseIncubation

0 to 4 °CElectroporationTemperature

Phosphate Buffered Saline (PBS),pH 7.4


5x10 (6) cells / 700 µlCell Density

700 µlVolume of Cells

20 to100 µg / 700 µlDNA Concentration

PBS or TE (10 mM Tris, 1 mM EDTA, pH8.0)


20 µl to 50 µl; (conc.=1µg / µl)Volume of DNA


0.4 cmCuvette Gap

0.75 kVVoltage


25 µFCapacitor



Hams F12 (Gibco)Outgrowth Medium


2 days before selectionLength of Incubation

F12 - 5158 plus or minus MTXSelection Methodor Assay Used

250 to1000 transformants / µg DNAElectroporationEfficiency

25%Per Cent Survival

Lars Adamson M.ScName of Submittor

Kabigen ABCell BiologyStrandbergsgatan 49S-11287 Stockholm, Sweden

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl, 0.2g KCl,0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES,pH 7.2, 150 mM NaCl, 5 mM CaCl2


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Medium*


089

Survey Number


Hamster, CHO, ovarySpeciesUsed

DNA: plasmids of 8.4 kb, linearizedMoleculesElectroporated

DMEM, 10% Fetal Bovine Serum, NEAA,Pennicillin /Streptomycin

Cell Growth Medium LogGrowth Phase atHarvest

Berg buffer (see Ref in notes)Wash Solution

10 min on icePre-pulseIncubation


Berg buffer (see Ref. in notes)ElectroporationMedium

10(8) cells /0.8ml Cell Density


20 µgDNA Concentration

waterDNA ResuspensionBuffer

20 µlVolume of DNA

Gene Pulser® Instruments Used

0.4 cmCuvette Gap

0 .80 kVVoltage


25 µFCapacitor


0 .6 to 0.8 msecTime Constant

DMEM, 10% FBS, NEAA, Pen/StrepOutgrowth Medium


2 weeksLength of Incubation

G418, 8-Aza adenineSelection Methodor Assay Used

10 transfectants / µg DNAElectroporationEfficiency

20 to 80% (varies)Per Cent Survival

Not givenName of Submittor

Not givenInstitutionAddress

Note: exponential values designated in parentheses.Ref: Chu,G., Hayakawa, H. and Berg, P.NAR 15 , 1131.Berg buffer: 20 mM HEPES, pH 7.05,137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose.


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Medium*


090

Survey Number



Proteins: restriction enzymes (Eco RI,ScaI, Dra I) and catalase


McCoys 5a (GIBCO/BRL, Sigma) Cell Growth Medium ExponentialGrowth Phase atHarvest

Phosphate Buffered Saline (PBS) or serum-free mediumWash Solution

Hepes Buffered Saline (HBS)Pre-pulseIncubation

4°CElectroporation

Temperature

HEPES Buffered Saline (HBS)ElectroporationMedium

2 x 10 (6) cells / mlCell Density


Not givenDNA Concentration




0.4 cmCuvette Gap

0.3 kVVoltage


960 µFCapacitor



McCoys 5AOutgrowth Medium


20 hr.Length of Incubation

Not givenSelection Methodor Assay Used


40 to 60%Per Cent Survival

Dr. Felipe CortesName of Submittor

Faculty of BiologyCell Biology Avenida Reina Mercedes s.n.E-41012 SevillaSpain

InstitutionAddress

Note: exponential values designated in parentheses.Cortez, F., and Ortiz,T.Mutation Research 246(1):221-6PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


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Medium*


091

Survey Number



DNA: multiple CAT and other vectorsunder different promoters includingHIV,SIV, human and rhesuscytomegalovirus immediate early gene,SV40 and the simian retrovirus type 2.


RPMI 1640 with 10 mM dextrose and0.1mM dithiothreitol (Sigma, GIBCO/ BRL,Flow Labs)

Cell Growth Medium Log phase (We routinely subdivide cells 24hours prior to electroporation)

Growth Phase atHarvest

Trypsin harvest, washed twice in Phosphate Buffered SalineWash Solution

NonePre-pulseIncubation

25 °C during and after electroporationElectroporation

Temperature

RPMI 1640 without Fetal Calf Serum ,+10mM dextrose, 0.1 mM dithiothreitol


1.3 x 10 (7) viable cells / ml, 0.3 mlCell Density


500 µg / ml (5 to 10 µg per pulse)DNA Concentration


10 to 20 µlVolume of DNA


0.4 cmCuvette Gap

0.25 kVVoltage


960 µFCapacitor



DMEM culture media, 10% fetal calf serumOutgrowth Medium


48 hoursLength of Incubation

Transient (CAT, b-gal, immunohisto- chemistry)Selection Methodor Assay Used

50 to100% ElectroporationEfficiency


Peter Barry, Ph.D., Asst. Research VirologistName of Submittor

Univ. of California- DavisDepartment of Medical Pathology , MS1ADavis, CA 95616

InstitutionAddress

Note: exponential values designated in parentheses.All plasmids were prepared by alkaline lysis and banded toequilibrium twice in cesium chloride gradients. Largest DNAmolecule electroporated has been the rhesus cytomegalovirusgenome (approx. 225 kilobases). In general, the higher theefficiency of transfection, the higher degree of cell killing. Forsome cell types,> 90% of the input cells are killed, although >90% of the survivors express very high levels of input DNA. Wehave found that ≥ 20 µg of DNA per electroporation resulted ingreatly reduced efficiency of transfection. Similarly, 2.5 µg ofDNA resulted in reduced efiiciency.


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After the Pulse



Medium*


092

Survey Number


Human, CEMx174, T lymphoblastoid SpeciesUsed

















0.4 cmCuvette Gap

0.200 kVVoltage


960 µFCapacitor



DMEM culture media, 10% Fetal Calf SerumOutgrowth Medium



Transient (CAT, b-gal, immunohisto- chemistry)Selection Methodor Assay Used

50 to 100% ElectroporationEfficiency




InstitutionAddress

Note: exponential values designated in parentheses.All plasmids were prepared by alkaline lysis and banded toequilibrium twice in cesium chloride gradients. Largest DNAmolecule electroporated has been the rhesus cytomegalovirusgenome (approx. 225 kilobases). In general, the higher theefficiency of transfection, the higher degree of cell killing. Forsome cell types,> 90% of the input cells are killed, although >90% of the survivors express very high levels of input DNA. Wehave found that ≥ 20 µg of DNA per electroporation resulted ingreatly reduced efficiency of transfection. Similarly, 2.5 µg ofDNA resulted in reduced efficiency.


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Medium*


093

Survey Number


Human, GCT, fibrous histocytoma, metastasis to lungSpeciesUsed

















0.4 cmCuvette Gap

0.20 kVVoltage


960 µFCapacitor

(Pulse Controller) Ω noneResistor





Transient (CAT, β-gal, immunohisto- chemistry).Selection Methodor Assay Used





InstitutionAddress

Note: exponential values designated in parentheses.All plasmids were prepared by alkaline lysis and banded toequilibrium twice in cesium chloride gradients. Largest DNAmolecule electroporated has been the rhesus cytomegalovirusgenome (approximately 225 kilobases). In general, the higher theefficiency of transfection, the higher degree of cell killing. Forsome cell types, > 90% of the input cells are killed, although >90% of the survivors express very high levels of input DNA. Wehave found that ≥ 20 µg of DNA per electroporation resulted ingreatly reduced efficiency of transfection. Similarly, 2.5 µg ofDNA resulted in reduced efficiency.


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Medium*


094

Survey Number


Human, HeLa, epithelial carcinomaSpeciesUsed

DNA: multiple CAT and other vectorsunder different promoters including HIV,SIV, human and rhesus cytomegalovirusimmediate early gene, SV40 and thesimian retrovirus type 2.










1.3 x 10 (7) viable cells / ml, used 0.3 mlCell Density






0.4 cmCuvette Gap

0.300 kVVoltage


500 µFCapacitor






Transient (CAT, b-gal, immunohisto-chemistry).Selection Methodor Assay Used





InstitutionAddress

Note: exponential values designated in parentheses.All plasmids were prepared by alkaline lysis and banded toequilibrium twice in cesium chloride gradients. Largest DNAmolecule electroporated has been the rhesus cytomegalovirusgenome (approx. 225 kB). In general, the higher the efficiency oftransfection, the higher degree of cell killing. For some cell types,> 90% of the input cells are killed, although > 90% of the survivorsexpress very high levels of input DNA. We have found that ≥ 20 µgof DNA per electroporation resulted in greatly reduced efficiencyof transfection. Similarly, 2.5 µg of DNA resulted in reducedefficiency.


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Medium*


095

Survey Number



DNA: plasmidMoleculesElectroporated

Not givenCell Growth Medium 50 to 80%Growth Phase atHarvest

Not givenWash Solution

Ice, 10 minutesPre-pulseIncubation


Temperature


1 x 10 (7 ) cells / mlCell Density


1 µg / mlDNA Concentration

TE Buffer (10 mM Tris, 1 mM EDTA)DNA ResuspensionBuffer

5 µlVolume of DNA

Not givenInstruments Used

0.4 cmCuvette Gap

0.25 kVVoltage


960 µFCapacitor

(Pulse Controller) none ΩResistor


Not givenOutgrowth Medium

Not givenOutgrowth Temperature

Not givenLength of Incubation



Not givenPer Cent Survival

Phang Seng Meng, Graduate StudentName of Submittor

Dr. Thomas Leong LaboratoryNational University of SingaporeInstitute Molecular Cell & BiologyKent Ridge CrecentSingapore 0511

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4,1.15g Na2HPO4


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Medium*


096

Survey Number

Mammalian, suspensionCell Type


DNA: pBLCAT2 series, 4.5 kBMoleculesElectroporated

MEM + 10% Fetal Calf Serum(GIBCO/BRL, Sigma)



5 minutes on icePre-pulseIncubation

4 °C (from ice to chamber at 25 °C )Electroporation

Temperature


2 x 10 (6) cells in cuvetteCell Density


2 to 20 µgDNA Concentration

TE Buffer (10 mM Tris, 1 mM EDTA)DNA ResuspensionBuffer



0.4 cmCuvette Gap

0.250 kVVoltage


960 µFCapacitor



MEM + 10% Fetal Calf SerumOutgrowth Medium



CAT assaySelection Methodor Assay Used

Not determinedElectroporationEfficiency


Mr. Terence ChongName of Submittor

National University of SingaporeInstitute Molecular Cell & BiologyKent Ridge CrecentSingapore 0511

InstitutionAddress


Nucleic Acid Research,18(3): 465-470 (1990).PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4,1.15g Na2HPO4


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Before the Pulse

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Medium*


097

Survey Number





DMEM, 10% Fetal Calf Serum [FCS]

(GIBCO/BRL, Sigma)



4° C, 10 min.Pre-pulseIncubation


Temperature

HEPES Buffered Saline, 6mM glucoseElectroporationMedium

5 x 10(6) cells/pulse for transient assay;Cell Density



Not given; pulse volume: 0.8 mlDNA ResuspensionBuffer

Not given; pulse volume: 0.8 mlVolume of DNA


0.4 cmCuvette Gap

0.170 kVVoltage


960 µFCapacitor


30 msecTime Constant

DMEM, 10% Fetal Calf Serum (FCS)Outgrowth Medium



G418 (stable transfections) andtransient assays






InstitutionAddress


Note: Stable transfections generally do not use carrier DNA.Also, the level of selective agent required to kill offnon-transfected cellsneeds to be established before transfection.The level required should kill non-transfected cells inapproximately 7 days.

HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


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Before the Pulse

After the Pulse



Medium*


098

Survey Number


Human, Hep3b2, hepatocytes; HeLa, epithelial carcinomaSpeciesUsed

DNA: various plasmids, about 3kB.MoleculesElectroporated

DMEM, MAB87 /3 + 10% serum(GIBCO/BRL, Sigma)

Cell Growth Medium Not givenGrowth Phase atHarvest


5 min.Pre-pulseIncubation


Cell growth mediumElectroporationMedium

5 x 10 (6) cells / pulseCell Density



Sterile deionized waterDNA ResuspensionBuffer

5 to10 µlVolume of DNA


0.4 cmCuvette Gap

0. 220 kVVoltage


960 µFCapacitor



Same as growth mediumOutgrowth Medium


OvernightLength of Incubation

b-gal staining (X-gal)luciferase


52%ElectroporationEfficiency


Margie Wilde, Research AssistantName of Submittor

Texas Children's HospitalPathology Department6621 FanninHouston, Texas 77030

InstitutionAddress


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Medium*


099

Survey Number


Human, HepG2, hepatomaSpeciesUsed

DNA: supercoiled DNA used for transienttransfections.


DMEM, 10% Fetal Calf Serum (FCS)(GIBCO/BRL, Sigma)


Wash two times in electroporation buffer.Wash Solution


Pre-pulseIncubation


Temperature

HEPES Buffered Saline, 6mM glucose,(optional: add 50 µl FCS, 50 µl salmonsperm DNA).








0.4 cmCuvette Gap

0.220 kVVoltage


960 µFCapacitor



F12, 10% Fetal Calf Serum (FCS)Outgrowth Medium



Transient assaysSelection Methodor Assay Used





InstitutionAddress


HBS: 10mM HEPES, pH 7.2, 150 mM NaCl,5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


100

Survey Number


Human, HuT78, cutaneous T-cell lymphomaSpeciesUsed

DNA: multiple CAT and other vectorsunder different promoters including HIV,SIV, human and rhesus cyto-megalovirus immediate early gene, SV40and the simian retrovirus type 2.










1.3 x 10 (7) viable cells / mlCell Density






0.4 cmCuvette Gap

0.250 kVVoltage


960 µFCapacitor






Transient (CAT, b-gal, immunohisto- chemistry).Selection Methodor Assay Used





InstitutionAddress



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Medium*


101

Survey Number


Human, JEG-3, choriocarcinoma cellsSpeciesUsed

DNA: growth hormone reporter constructwith a mammalian promoter.


MEM + 10% Fetal Bovine Serum + 1%glutamine + 1% penicillin / streptomycin(GIBCO/BRL, Sigma)

Cell Growth Medium 70% confluencyGrowth Phase atHarvest

MEM + 10% Fetal Bovine SerumWash Solution

Not givenPre-pulseIncubation

0 °C ( ice)Electroporation

Temperature

MEM + 10% FBSElectroporationMedium

2.5 x 10 (8) cells / mlCell Density


4 µg / µlDNA Concentration

TE Buffer (10 mM Tris, 1 mM EDTA, pH8.0)


12 µlVolume of DNA


0.4cmCuvette Gap

0.140 kVVoltage


960 µFCapacitor




ice °COutgrowth Temperature

10 minLength of Incubation

Growth hormone RIA or ELISASelection Methodor Assay Used


50 %Per Cent Survival

Rich Bond, Principal ScientistName of Submittor

Schering ResearchMolecular Pharmacology Bldg. B-9-160 Orange St.Bloomfield, NJ 07003

InstitutionAddress


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Medium*


102

Survey Number


Human, JY cell (human B cell, Epstein-Barr virus transformed)SpeciesUsed

DNA, plasmid (10 kB)MoleculesElectroporated

DMEM (GIBCO/BRL, Sigma)Cell Growth Medium Not givenGrowth Phase atHarvest




Temperature

Not givenElectroporationMedium

2 x 10 (7) cells / 0.8 mlCell Density

Not givenVolume of Cells





0.4 cmCuvette Gap

Not given Voltage

Not given Field Strength

Not given Capacitor


Not given Time Constant

DMEMOutgrowth Medium


10 min.Length of Incubation


5 x 10 (5) transfectants / µg DNAElectroporationEfficiency

1%Per Cent Survival

Mr. Junji TakedaName of Submittor

Osaka UniversityMicrophysiology SectionImmun. Deficiency ResearchJapan

InstitutionAddress


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Before the Pulse

After the Pulse



Medium*


103

Survey Number


Human, K562, chronic myeloid leukemiaSpeciesUsed

DNA: pRSVneo, linearized, 5.6 kBMoleculesElectroporated

RPMI + 20% Fetal Calf Serum(GIBCO/BRL,Sigma)


RPMIWash Solution



Temperature

RPMIElectroporationMedium

5 x10 (6) cells / mlCell Density

0.4 to 0.8 mlVolume of Cells

5 µg / 800 µlDNA Concentration


up to 50 µlVolume of DNA


0.4cmCuvette Gap

up to 2 kVVoltage

up to 5 kV/cmField Strength

up to 960 µFCapacitor



RPMI + 20% Fetal Calf SerumOutgrowth Medium


48 hr.Length of Incubation

G418Selection Methodor Assay Used

0.3% clonogenic cells / µgElectroporationEfficiency


Ms. Genevieve M. CroakerName of Submittor

Royal Prince Alfred HospitalKanematsu LaboratoriesMissenden RoadCamperdown, NSW 2050Australia

InstitutionAddress


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Medium*


104

Survey Number

MammalianCell Type

Human, K562, chronic myeloid leukemiaSpeciesUsed

DNA: pZ189MoleculesElectroporated

RPMI 1640 (GIBCO/BRL,Sigma)Cell Growth Medium 10 (6) cells / mlGrowth Phase atHarvest



20 to 25 °C (but pre-cooled on ice)Electroporation

Temperature





TEDNA ResuspensionBuffer

40 µgVolume of DNA


0.4 cmCuvette Gap

0.6 kVVoltage

1.50 V/cmField Strength

25 µFCapacitor


1.5 to 1.9 msecTime Constant

RPMI 1640Outgrowth Medium

37 °C, 5% CO2Outgrowth Temperature

1 dayLength of Incubation




Fumio Yatagai, ScientistName of Submittor

RIKEN (The Inst. of Phy. & Chem. Res.)Radiation Biology2-1 HirosawaWako-shi, Saitama 351-01JAPAN

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 1.15g Na2HPO4


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Before the Pulse

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Medium*


105

Survey Number


Human, K562, chronic myeloid leukemia.SpeciesUsed

DNA: linearized (5kB) n-rasCAT plasmidsMoleculesElectroporated

RPMI + 20% Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium log phaseGrowth Phase atHarvest

RPMIWash Solution



Temperature


10 (7) cells / cuvetteCell Density


50 µg DNA / cuvetteDNA Concentration




0.4 cmCuvette Gap

2.0 kVVoltage

5 kV/cmField Strength

25 µFCapacitor



RPMI + 20% Fetal Calf SerumOutgrowth Medium






Ms. Jacqueline ThornName of Submittor

Royal Prince Alfred HospitalMissenden Road,Camperdown, NSW 2050AUSTRALIA

InstitutionAddress


It is NOT RECOMMENDED to use high voltage without the PulseController.


The Pulse

Before the Pulse

After the Pulse



Medium*


106

Survey Number


Human, K562, chronic myeloid leukemia; U937, histiocyticlymphoma

SpeciesUsed

DNA: linearized plasmid DNA; RSV LTRpromoting human HLA Class II genes,along with HTK promoter(neo r gene) containing plasmid(co-transfection)


RPMI 1640 + 10% Fetal Calf Serum(GIBCO/ BRL, Sigma)

Cell Growth Medium Early log phase growthGrowth Phase atHarvest

RPMI without serumWash Solution



RPMI without serumElectroporationMedium

10 (7) cells / mlCell Density





20 µlVolume of DNA


0.4 cmCuvette Gap

0.2 kVVoltage


960 µFCapacitor



RPMI 1640 + 10% Fetal Calf SerumOutgrowth Medium


15 to 30 daysLength of Incubation

G418 resistanceSelection Methodor Assay Used

Unknown (see notes)ElectroporationEfficiency

<1%Per Cent Survival

W. Scott GoebalName of Submittor

Indiana Univ. School of MedicineRiley Hospital Room S-09702 Barnhill Dr.Indianapolis, IN 46202-5200

InstitutionAddress

Note: exponential values designated in parentheses.Cells resistant to G418 (1µg/ml) grew out of post-pulse cellculture after about 15-30 days, with low % of survival. G418resistant cells were then dilution-cloned in preparation forscreening a Class II expressing clone. However, before themajority of cells could be screened, the clones were lost. I don'tknow how efficient electroporation procedure was since we weretrying to get expression of a two-chain molecule on the cellsurface - many factors could go wrong post-electroporation butprior to protein expression on the cell surface.


The Pulse

Before the Pulse

After the Pulse



Medium*


107

Survey Number


Human, Raji, Burkitt lymphoma; Jurkat, acute T cell leukemia;T-cells

SpeciesUsed

DNA: linear, 5 kBMoleculesElectroporated

RPMI 1640 + 10% Fetal Calf Serum(GIBCO/BRL, Sigma)


Hepes Buffered SalineWash Solution





0.5 to 0.75 mlVolume of Cells



11 µlVolume of DNA


0.4 cmCuvette Gap

0.40 kVVoltage


960 µFCapacitor


Not givenTime Constant




Preparation of RNA or CAT assaySelection Methodor Assay Used

Not quantifiedElectroporationEfficiency


Jeremy M. Boss, Ph.D. Asst. ProfName of Submittor

Emory UniversityMicrobiology / ImmunologyRollins Research CenterAtlanta, GA 03022

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


108

Survey Number


Human, 293s, transformed primary embryonic kidney; Lymphoblastcell lines, EBV immortilized

SpeciesUsed

DNA, p220LTR, 11.2 kb, supercoiled.MoleculesElectroporated

RPMI,10% Fetal Calf Serum,Penicillin,Streptomycin, L-Glutamine, orDMEM (GIBCO/BRL, Sigma)

Cell Growth Medium 80% confluent or 1 x 10 (6) cells / ml Growth Phase atHarvest

Electroporation Buffer (see notes)Wash Solution



See notesElectroporationMedium

1 x 10 (7) cells / µlCell Density


1 to 4 µg / µlDNA Concentration



<20 µlVolume of DNA


0.4 cm Cuvette Gap

0.260 kV Voltage

0.65 kV/cm Field Strength

550 or 960 µFCapacitor


6.4 or 11.2 msec Time Constant

RPMI,10% Fetal Calf Serum,Penicillin,Streptomycin, L-Glutamine, or DMEM

Outgrowth Medium

37°COutgrowth Temperature

InfiniteLength of Incubation

Hygromycin resistanceSelection Methodor Assay Used

4000 tranformants / µg DNAElectroporationEfficiency


David Van Der BergName of Submittor

Stanford UniversityGenetics DepartmentSchool of Medicine, S-337Stanford, CA 94305

InstitutionAddress


* Electroporation buffer from Chu,et. al.,NAR 15 (3): 1311 (1987);1x HeBS, 20 mM HEPES, pH 7.05,137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose.


The Pulse

Before the Pulse

After the Pulse



Medium*


109

Survey Number


Mouse, L929, connective tissue, clone of strain LSpeciesUsed









RPMI 1640 without Fetal Calf Serum,+10mM dextrose, 0.1 mM dithiothreitol








0.4 cmCuvette Gap

0.350 kVVoltage


500 µFCapacitor











InstitutionAddress



The Pulse

Before the Pulse

After the Pulse



Medium*


110

Survey Number


Human, MCF-7, breastSpeciesUsed



RPMI, 10% Fetal Calf Serum (FCS), insulin10µg / ml (GIBCO/BRL, Sigma)



4° C, 10 min. (optional: add 50 µlFCS if using HeBS aselectroporation media; 50 µlsalmon sperm DNA for transienttransfections)

Pre-pulseIncubation


Temperature



5 x 10 (6) cells/pulseCell Density



Not given; final pulse volume: 0.8 mlDNA ResuspensionBuffer

Not given; final pulse volume: 0.8 mlVolume of DNA


0.4 cmCuvette Gap



960 µFCapacitor



RPMI, 10% Fetal Calf Serum (FCS),insulin,10µg/ml

Outgrowth Medium








InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


111

Survey Number


Human, MRC-5, lung, diploidSpeciesUsed

















0.4 cmCuvette Gap

0.3 kVVoltage


960 µFCapacitor











InstitutionAddress



The Pulse

Before the Pulse

After the Pulse



Medium*


112

Survey Number


Human, MRC-5 / V1, lung fibroblasts, transformedSpeciesUsed

DNA: neo gene.MoleculesElectroporated

DMEM + 10% Fetal Calf Serum(GIBCO/BRL, Sigma)



37° C, 1/2 hourPre-pulseIncubation


DMEMElectroporationMedium

5 x 10 (6) cellsCell Density

1.4 ml (**see notes)Volume of Cells



20 µlVolume of DNA

Gene Pulser® apparatusInstruments Used

0.4 cmCuvette Gap

2.0 kVVoltage


25 µFCapacitor

(Pulse Controller) Ω none; NOTResistor


DMEM, 10% Fetal Calf SerumOutgrowth Medium


1 month for selectionLength of Incubation

1 mg / ml geneticinSelection Methodor Assay Used


Not given Per Cent Survival

Dr. Ann M. SimpsonName of Submittor

University of SydneyMedicine DeptN.S.W. 2006Sydney, AUSTRALIA

InstitutionAddress


**It is NOT RECOMMENDED to use high voltage with out thePulse Controller. It is not recommended to use more than 0.8 mlin the 0.4 cm cuvette; greater volumes may create non-uniformfield strengths during the pulse.


The Pulse

Before the Pulse

After the Pulse



Medium*


113

Survey Number


Human, pancreatic cell linesSpeciesUsed

DNA: pSU2 neo, PAG 60MoleculesElectroporated

Daigo's T (10% Fetal Calf Serum)Cell Growth Medium Not givenGrowth Phase atHarvest

Hanks' Balanced Salt SolutionWash Solution



Temperature

Hanks' Balanced Salt SolutionElectroporationMedium

1.5 x 10 (6) / mlCell Density

1 mlVolume of Cells

1 to 10 µg / mlDNA Concentration

Hanks' Balanced Salt Solution DNA ResuspensionBuffer

1 mlVolume of DNA

Gene Pulser ® apparatusInstruments Used

Not given Cuvette Gap


Not givenField Strength

25 µFCapacitor



Daigo's T (5% FCS)Outgrowth Medium


not givenLength of Incubation

pSV2 Neo (G418)Selection Methodor Assay Used

1 to 20 clones /1µl DNA; depends on the cell lineElectroporationEfficiency


Akira Kono, Ph.D.Name of Submittor

Research Institute, Kyushu Cancer CenterDivision of Chrmotherapy3-1-1 Notamo, Minami-kuFukuoka, 815 JAPAN

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


114

Survey Number


Human, red blood cellsSpeciesUsed

Dextrans, various proteinsMoleculesElectroporated

Harvested from whole bloodCell Growth Medium Not givenGrowth Phase atHarvest

Isotonic Phosphate Buffered Saline(PBS)

Wash Solution

Held on ice in Phosphate BufferedSaline

Pre-pulseIncubation

0 or 25 °CElectroporationTemperature

20 mM Phosphate Buffered SalineElectroporationMedium

10 (6) to 10 (7) cells / mlCell Density


10 (-5) to 10 (-4) M dextranDNA Concentration

Protein in Phosphate Buffered SalineDNA ResuspensionBuffer



0.2 cmCuvette Gap

0 to 2.5 kVVoltage

0 to 12.5 kV/cmField Strength

25 µFCapacitor

(Pulse Controller) not used.Resistor


Phosphate Buffered SalineOutgrowth Medium


0 to 5 hr.Length of Incubation

Flow cytometry, detection of fluorescentmolecules


up to 90% of cells exhibit uptakeElectroporationEfficiency


Mark PrausnitzName of Submittor

MITChemical EngineeringRoom E25-342, 77 Massachusetts AvenueCambridge, MA 02139

InstitutionAddress


**It is NOT RECOMMENDED to use high voltage with out thePulse Controller.PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 1.15g Na2HPO4


The Pulse

Before the Pulse

After the Pulse



Medium*


115

Survey Number


Human, U373, glioblastoma, astrocytoma, grade III SpeciesUsed




Cell Growth Medium Log phase (We routinely subdivide cells 24hours prior to electroporation













0.4 cmCuvette Gap

0.3 kVVoltage


960 µFCapacitor











InstitutionAddress



The Pulse

Before the Pulse

After the Pulse



Medium*


116

Survey Number


Human,U937, histiocytic lymphoma SpeciesUsed

DNA: plasmidMoleculesElectroporated

RPMI 1640, 10 % Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium 5 to 10 x10 (5) / mlGrowth Phase atHarvest


Phosphate Buffered Saline (-)Pre-pulseIncubation


Temperature


1 x 10 (8) / mlCell Density

2 x 10 (7) / 200 µlVolume of Cells

50 µg / 200 µlDNA Concentration


50 µgVolume of DNA


0.4 cmCuvette Gap

0.2 kVVoltage


960 µFCapacitor



RPMI 1640, 10% Fetal Calf SerumOutgrowth Medium


1 dayLength of Incubation

G418: 700 µg / ml 3 days; then400 µg / ml 3 days.


Not assayedElectroporationEfficiency

Not assayedPer Cent Survival

Shumsuke MoriName of Submittor

Institute for Medical GeneticsExperimental GeneticsKuhanji 4-24-1Kumamoto, JAPAN

InstitutionAddress

Note : exponential values designated in parentheses.Relevant Publications and/or Comments

The Pulse

Before the Pulse

After the Pulse



Medium*


117

Survey Number


Human, U937, hystiocytic lymphoma SpeciesUsed

DNA: plasmid,closed circular,CsClpurified x2; Co-transfection with 2plasmids; pHIV LTR-CAT andpSV40-Tat; also transfection withpCH110 alone (7.2 kB).


RPMI 1640 + 10% fetal calf serum +penicillin/streptomycin, L-glutamine,sodium pyruvate (GIBCO/BRL, Sigma)

Cell Growth Medium Log growthGrowth Phase atHarvest

Cell growth mediaWash Solution

10 min at 4° C.Pre-pulseIncubation


Cell growth mediaElectroporationMedium

1.3 x 10 (7) / mlCell Density






0.4 cmCuvette Gap

0.30 kVVoltage


960 µFCapacitor



Cell growth mediaOutgrowth Medium


24 to 48 hr.Length of Incubation

CAT assay, stain for β-galactosidaseSelection Methodor Assay Used

About 500 transfectants / µg DNAElectroporationEfficiency


Michael S. Bernstein, M.D. Asst. Prof.Name of Submittor

Univ of California, San FranciscoDept. of MedicineBox 0654San Francisco, CA 94143

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


118

Survey Number


Human, UC729-6, lymphoblastoid, B-cellsSpeciesUsed

DNA: EBV based vectors, 12-20 kB,closed circular


RPMI 1640Cell Growth Medium Mid log at 1 x 10 (6) cells / mlGrowth Phase atHarvest

Phosphate Buffered Saline (PBS)Wash Solution

Hepes buffered saline (HBS)Pre-pulseIncubation


Temperature

Hepes Buffered Saline (HBS)ElectroporationMedium


1.0 ml total volume (see notes)*Volume of Cells

500 µg / ml totalDNA Concentration

450 µg / ml carrier + 50 µg / ml plasmidDNA ResuspensionBuffer



0.4 cmCuvette Gap

0.32 kVVoltage


960 µFCapacitor



RPMI 1640Outgrowth Medium



HygromycinSelection Methodor Assay Used

2 x 10 (4) to 5 x 10 (4)ElectroporationEfficiency


Robert MargolskeeName of Submittor

Roche Institute of Molecular BiologyNeuroscienceNutley, NJ 07110

InstitutionAddress

Note: exponential values designated in parentheses.(1.) Margolshee et. al., Mol. Cell. Biol. 8: 2837-2847 (1988). (2.)Canfield et. al ., Mol. Cell. Biol. 10: 1367-1372 (1990). (3.)Spickofsky et. al., DNA and Prot. Eng. Techniques , 2: 14-18 (1990).*Maximum recommended volume for 0.4 cm cuvettes is 0.8 mlfor uniform field strengths.PBS: 1x = 8g NaCl ,0.2g KCl, 0.2g KH2PO4,1.15g Na2HPO4HBS: 10mM HEPES, pH 7.2, 150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


119

Survey Number

MammalianCell Type

Human, V79, skin cells, fibroblastsSpeciesUsed

DNA: plasmid.MoleculesElectroporated

Modified MEM, 5%Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium Actively growing 70% confluentGrowth Phase atHarvest


10 min / icePre-pulseIncubation


Temperature

10 mM HEPESElectroporationMedium

10 (7) cells / ml Cell Density




10 µlVolume of DNA


0.4 cmCuvette Gap

0.450 kVVoltage


25 µFCapacitor

5 ΩResistor




1 weekLength of Incubation

G418 or ampicillinSelection Methodor Assay Used

10 (6) transformants / µgElectroporationEfficiency

2%Per Cent Survival

Phillip K. Liu, Ph.D., Assistant ProfessorName of Submittor

M.D. Anderson Cancer CenterLaboratory Medicine Box 721515 Halcombe Blvd.Houston, Texas 77030

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


120

Survey Number

MammalianCell Type

Mouse, A-9, derivative of mouse L cell (contains humanchromosomes)

SpeciesUsed

DNA, various linear constructs of humansequences & selectable markers.


DMEM (GIBCO/BRL, Sigma)Cell Growth Medium Not givenGrowth Phase atHarvest

Phosphate Buffered Saline, without Ca++, Mg++Wash Solution

10 min. icePre-pulseIncubation

Cuvette on ice just prior to pulseElectroporation

Temperature

Phosphate Buffered Saline, without Ca++,Mg++




1 µg /1 µlDNA Concentration


10 µlVolume of DNA


0.4 cmCuvette Gap

0.45 kVVoltage


500 µF and 960 µFCapacitor






Hygromycin, G418, or MXSelection Methodor Assay Used

LowElectroporationEfficiency


Keith RosenbachName of Submittor

UMDNJMicrobiology Department185 South Orange AveNewark, NJ 07103

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


121

Survey Number


B-cell line, unspecified, and heteromyelomaSpeciesUsed

ElectrofusionMoleculesElectroporated

RPMI+ 10% Fetal Calf Serum+ 2 mML-Glutamine (GIBCO/ BRL, Sigma)

Cell Growth Medium Exponential growthGrowth Phase atHarvest

RPMIWash Solution

NoPre-pulseIncubation


Temperature


10 (8) cells / µlCell Density




10 (8) cells / mlVolume of DNA


0.2 cmCuvette Gap

0.160 kVVoltage


960 µFCapacitor



HAT medium and azaserine & ouabainOutgrowth Medium





No fusionPer Cent Survival

Berta Sanchez, Ph.D.Name of Submittor

Hospital Virgen Del RocioImmunolociaAvda Manuez Siurot s/n41013 SevillaSpain

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


122

Survey Number


Mouse, BALB/c 3T3, clone A31, fibroblast, whole embryo / fetus,normal

SpeciesUsed




Cell Growth Medium Log phase. We routinely subdivide cells 24hours prior to electroporation.





RPMI 1640 without Fetal Calf Serum,+10mM dextrose,0.1 mM dithiothreitol








0.4 cmCuvette Gap

0.4 kVVoltage


500 µFCapacitor






Transient (CAT, β-gal, immunohisto-chemistry).Selection Methodor Assay Used





InstitutionAddress



The Pulse

Before the Pulse

After the Pulse



Medium*


123

Survey Number


Mouse, C2 cell line, muscle myoblastSpeciesUsed

DNA: p1481D (retroviral),11 kB,supercoiled.


DMEM (GIBCO/ BRL, Sigma)Cell Growth Medium log phaseGrowth Phase atHarvest

Hepes buffered sucrose, PBS, and phosphate buffered sucroseWash Solution


4 °C or Room temperatureElectroporation

Temperature

Phosphate or HEPES buffered sucroseElectroporationMedium

variedCell Density

200 µl to 800 µlVolume of Cells

1 µg to 10 µgDNA Concentration




0.1 cmCuvette Gap

1.0 kVVoltage


1 µFCapacitor

(Pulse Controller ) infinity setting Resistor

0 .3 to 0.4 msecTime Constant



24 to 48 hoursLength of Incubation

X-gal stain for LacZSelection Methodor Assay Used

1.5 x 10 (3) transformants / µg DNAElectroporationEfficiency

Not knownPer Cent Survival

Barb Rainish - Graduate StudentName of Submittor

University of IllinoisBiochemistry / Cell Biology505 S. Goodwin - 506 Morrill HallUrbana, IL 61801

InstitutionAddress

Note: exponential values designated in parentheses.PB Sucrose: 272 mM sucrose, 7 mM potassium phosphate, pH7.4, 1 mM MgCl2.HEPES Buffered Sucrose: 272 mM sucrose, 8 mM HEPES, pH7.4.


The Pulse

Before the Pulse

After the Pulse



Medium*


124

Survey Number


Mouse, C2C12, muscleSpeciesUsed



DMEM, 20% Fetal Calf Serum (FCS)Cell Growth Medium 50 to70% confluentGrowth Phase atHarvest


4° C, 10 min. (optional: add 50 µlFCS if using HeBS aselectroporation media; 50 µlsalmon sperm DNA for transienttransfections)

Pre-pulseIncubation


Temperature

HEPES Buffered Saline, 6mM glucose,(optional: 50 µl salmon sperm DNA).








0.4 cmCuvette Gap

0.220 kVVoltage


960 µFCapacitor



DMEM, 20% Fetal Calf Serum (FCS) Outgrowth Medium








InstitutionAddress




The Pulse

Before the Pulse

After the Pulse



Medium*


125

Survey Number


Mouse, C127, fibroblast, mammary tumorSpeciesUsed

DNA: pC59, an E2 of BPV-1 expressionplasmid; p407, an E2 responsive CATplasmid.


DMEM + 10% Fetal Bovine Serum(GIBCO/BRL, Sigma)


1x Phosphate Buffered Saline, 3 times

Wash Solution

Room temperaturePre-pulseIncubation


Temperature

DMEM + 10% FBS + 5mM BES, pH 7.2, 50µg salmon sperm, carrier DNA


1 to 5 x 10 (6) cells / mlCell Density


5 to 10 µg DNA DNA Concentration

in 5 to 10 µl 10 mM Tris Buffer,pH 8.0




0.4 cmCuvette Gap

0.21 kVVoltage


960 µFCapacitor



DMEM, 10% Fetal Bovine SerumOutgrowth Medium






Shrikant Anant, Graduate StudentName of Submittor

Univ of Illinois at ChicagoGenetics Dept.808 S. Wood #1404Chicago, IL 60612

InstitutionAddress


Similar protocol has been published by another lab. Reference isgiven below:

Ustav, M. and Stenlund, A. (1991) EMBO J . 10(2): 449-457.


The Pulse

Before the Pulse

After the Pulse



Medium*


126

Survey Number


Monkey, COS-1, kidneySpeciesUsed







Pre-pulseIncubation


Temperature



5 x 10 (6) cells/pulseCell Density






0.4 cmCuvette Gap

0.3 kVVoltage


250 µFCapacitor

(Pulse Controller) Ω none. Resistor










InstitutionAddress


HBS: 10mM HEPES, pH 7.2,150 mM NaCl,5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


127

Survey Number


Monkey, COS-7, kidney, SV-40 transformedSpeciesUsed

DNA: CMV b-gal , 6 kB, supercoiledMoleculesElectroporated

DMEM + 10% Fetal Bovine Serum(GIBCO/BRL, Sigma)

Cell Growth Medium Log phase, 70 to 80% confluentGrowth Phase atHarvest

TrypsinizeWash Solution

Room temperaturePre-pulseIncubation


Temperature

DMEM + 10% Fetal Bovine Serum + 5mMBES, pH 7.2



10 µg / 250 µl DNAVolume of Cells

10 mM Tris, 1 mM EDTA, pH 8.0)DNA Concentration




0.4 cmCuvette Gap

0.170 kVVoltage


960 µFCapacitor




Room temperatureOutgrowth Temperature

10 min., then pellet& resuspend in DMEMLength of Incubation




Cheng-Ming ChiangName of Submittor

University of Rochester Medical CenterBiochemistry, Box 607601 Elmwood AveRochester, NY 14642

InstitutionAddress


See reference: Ustav, M., and Stenlund, A. 1991.EMBO J . 10(2):449-457.


The Pulse

Before the Pulse

After the Pulse



Medium*


128

Survey Number


Monkey, COS-7, kidney, SV-40 transformedSpeciesUsed

















0.4 cmCuvette Gap

0.30 kVVoltage


960 µFCapacitor











InstitutionAddress



The Pulse

Before the Pulse

After the Pulse



Medium*


129

Survey Number


Mouse, embryonic stem cellsSpeciesUsed

DNA: plasmid, 12 kBMoleculesElectroporated

DMEM + L Glutamine + Pen/Strep + FetalCalf Serum + mercaptoethanol +non-essential amino acids.(GIBCO/BRL, Sigma)

Cell Growth Medium Exponential growth phaseGrowth Phase atHarvest


Not incubated pre-pulsePre-pulseIncubation


Temperature


1.5 to 2.0 x 10 (7) cells / pulseCell Density



Phosphate Buffered SalineDNA ResuspensionBuffer

100 µg / pulseVolume of DNA


0.4 cmCuvette Gap

0.160 kVVoltage


960 µFCapacitor



DMEM + L Glutamine + Pen/Strep + Fetal CalfSerum + mercaptoethanol + non-essential aminoacids.

Outgrowth Medium


2 weeks and moreLength of Incubation



2.38 x 10 (-5)Per Cent Survival

Dr. K.S.E. CheahName of Submittor

Hong Kong UniversityBiochemistry DepartmentSassoon RoadHong Kong

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


130

Survey Number


Mouse, embryonic stem cellsSpeciesUsed

DNA: pNeo Xp 5.3TK, pHyg Xp5.3 TK,about 10 kB.


20% Fetal Calf Serum, DMEM + aminoacids, 2- mercaptoethanol, nucleosides.(GIBCO/ BRL, Sigma)



5 min., on icePre-pulseIncubation

4 to10 °CElectroporationTemperature




20 µg DNA/ pulseDNA Concentration


16 µlVolume of DNA


0.4cmCuvette Gap

0.28 kVVoltage


500 µFCapacitor






G418, GANCSelection Methodor Assay Used

20 to 30 transfectants / µg DNAElectroporationEfficiency


Kiyoji Tanaka, Associate ProfessorName of Submittor

Inst. Mol. Cell. Biol.Osaka University, Cell Biology1-3 Yamada okaSuita, Osaka, 656JAPAN

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


131

Survey Number


Mouse, erythroleukemia cellsSpeciesUsed

DNA: pSV2 neo, 5 kBMoleculesElectroporated

RPMI ,10% Fetal Calf Serum(GIBCO/BRL, Sigma)



10 to 20 min.Pre-pulseIncubation



10 (7) cells / 50 µlCell Density






0.4 cmCuvette Gap

0.4 kVVoltage


25 µFCapacitor



RPMI,10% Fetal Calf SerumOutgrowth Medium


10 to 30 minutesLength of Incubation


Low (**see notes)ElectroporationEfficiency


Iliana Coccia ReseName of Submittor

Instituto Superiore SanitaDept of VirologyVisle Regina Elena 299Rome , ITALY 00164

InstitutionAddress


**Note: Transfection efficiemnces ofthese cells may be increasedwith the use of the Capacitance Extender (providing longer timeconstants).


The Pulse

Before the Pulse

After the Pulse



Medium*


132

Survey Number


Mouse, erythroleukemia cellsSpeciesUsed

DNA: variety of constructs, supercoiled,usually <10 kB (some pUC based)


DMEM + 10% Fetal Calf Serum, 1 x glutamine,1 x penicillin / streptomycin(GIBCO/BRL, Sigma)


DMEM + 10% Fetal Calf Serum,1 x glutamine

Wash Solution

10 min., room temperaturePre-pulseIncubation


Temperature

DMEM + 1% Fetal Calf SerumElectroporationMedium



20 to 30 µg /100 µlDNA Concentration

10 mM Tris, pH 8.0, 1 mM EDTADNA ResuspensionBuffer

100 µlVolume of DNA


0.4 cmCuvette Gap

0.250 kVVoltage


960 µFCapacitor

(Pulse Controller ) Ω none Resistor

16 msec (some small variation)Time Constant

DMEM + 10% Fetal Calf Serum, 1 x glutamine, 1x penicillin / streptomycin

Outgrowth Medium






Deborah L. GalsonName of Submittor

Center for Cancer Research, E17-536Mass. Inst. of Technology40 Ames St.Cambridge, MA 02139

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


133

Survey Number


Mouse, FDC-PI, Il-3-dependent cell lineSpeciesUsed

DNA: 6.5 kB, linearized plamid.MoleculesElectroporated

RPMI 1640, 10% Fetal Calf Serum(GIBCO/BRL, Sigma)


HEPES buffered saline (see notes)Wash Solution

Ice, 10 min.Pre-pulseIncubation


HEPES buffered salineElectroporationMedium



1 mg / mlDNA Concentration



10 µl (10 µg) / pulseVolume of DNA


0.4 cmCuvette Gap

1.5 kVVoltage


25 µFCapacitor



RPMI 1640, 10% Fetal Calf SerumOutgrowth Medium



G418, 400 µg / mlSelection Methodor Assay Used

10 to 20 transfectants / µg DNAElectroporationEfficiency

60% at 10 min. after electroporation Per Cent Survival

Satoshi Takaki, M.D.Name of Submittor

Inst. for Med. ImmunologyKumamoto Univ. Medical SchoolDept. of Biology , 2-2-1 HonjoKumamoto, JAPAN 860

InstitutionAddress


Ref: EMBO J . 9 : 4367-4374 (1990) Molecular cloning andexpression of the murine interlenkin -5 receptor.HEPES Buffered Saline: 140 mM NaCl, 5 mM KCl,0.75 mM Na2HPO4, 6 mM dextrose, 25 mM HEPES,pH 7.2**It is NOT RECOMMENDED to use high voltage with out thePulse Controller.


The Pulse

Before the Pulse

After the Pulse



Medium*


134

Survey Number


Mouse, 32d, J558L, myelomaSpeciesUsed

DNA: linear and circular plasmids, 4-12kB in size


RPMI,10% Fetal Calf SerumGIBCO/BRL, Sigma)

Cell Growth Medium logGrowth Phase atHarvest

logWash Solution

10 minutes icePre-pulseIncubation

(ice) 0 °CElectroporationTemperature

RPMI, 10% Nu-serumElectroporationMedium

10 (7) cells / µlCell Density


1 mg / mlDNA Concentration





0.4 cmCuvette Gap



960 µFCapacitor






G418; Hygromycin BSelection Methodor Assay Used



Alan MatsumotoName of Submittor

Johns Hopkins University725 N. Wolfe St.Baltimore, MD 21205

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


135

Survey Number


Mouse, J558-L, myelomaSpeciesUsed

DNA: plasmids, 4.5 kB, supercoiled.MoleculesElectroporated

DMEM + 10% Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium Split on the previous dayGrowth Phase atHarvest

NoneWash Solution



DMEM + 10% Fetal Calf SerumElectroporationMedium

10 (7) cells in 300 µlCell Density


4 µg DNA per 10 (7) cells DNA Concentration


4 µlVolume of DNA


0.4 cmCuvette Gap

0.25 kVVoltage


960 µFCapacitor



DMEM + 10% Fetal Calf SerumOutgrowth Medium






Srikumar Chellappan, Ph.D. Rsch Assoc.Name of Submittor

Duke University Medical CenterMicrobiology and ImmunologyP.O. Box 3054Durham, NC 27710

InstitutionAddress


PNAS, 87: 5788 (1990).

Electroporation worked best for J558L cells - DEAE dextran didnot; neither did lipofection.


The Pulse

Before the Pulse

After the Pulse



Medium*


136

Survey Number


Mouse, LM(TK-), connective tissue [L-M (TK-)].SpeciesUsed

DNA: pSV2neo, Apa L1 digest.MoleculesElectroporated

Dulbecco's MEM (GIBCO/BRL, Sigma)Cell Growth Medium 50 to 75% confluentGrowth Phase atHarvest

Phosphate Buffered Saline, without Ca++ or Mg++Wash Solution



Phosphate Buffered Saline, without Ca++or Mg++




0.5 µg / mlDNA Concentration

Phosphate Buffered sucroseDNA ResuspensionBuffer

10 µlVolume of DNA


0.4 cmCuvette Gap

0.950 kVVoltage


25 µFCapacitor

(Pulse Controller) Ω none.Resistor

0.4msecTime Constant

Dulbecco's MEMOutgrowth Medium



G-418 , 400 µg / ml after 48 hours recoverySelection Methodor Assay Used

1 x 10 (-3) transformants / cellElectroporationEfficiency


Deborah J. Hunter, Graduate StudentName of Submittor

University of UtahDept. of BiologySalt Lake City, Utah 84112

InstitutionAddress


**It is NOT RECOMMENDED to use high voltage with out thePulse Controller.


The Pulse

Before the Pulse

After the Pulse



Medium*


137

Survey Number


Mouse, LM(TK-), connective tissue,[L-M (TK-)].

SpeciesUsed







Pre-pulseIncubation


Temperature









0.4 cmCuvette Gap

0.300 kVVoltage


960 µFCapacitor











InstitutionAddress


HBS: 10mM HEPES,pH 7.2, 150 mM NaCl,5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


138

Survey Number

Mammalian, suspension Cell Type

Mouse, WEHI-3B, myelomonocytic leukemia SpeciesUsed

DNA: fos, jun (insert sizes are 4.2 and 1.8kb using different vectors), linear


McCoy's 5A (GIBCO/BRL, Sigma)

Cell Growth Medium ExponentialGrowth Phase atHarvest


10 min at room temperaturePre-pulseIncubation


Temperature




15 µg /10 µgDNA Concentration


10 µlVolume of DNA


0.4 cmCuvette Gap

0.25 kVVoltage


500 µFCapacitor



McCoy's 5AOutgrowth Medium


2 weeksLength of Incubation

G-418Selection Methodor Assay Used

Not doneElectroporationEfficiency


Jianming Li, Postdoctoral AssociateName of Submittor

Yale University School of MedicinePharmacology333 Cedar StreetNew Haven, CT 06510

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


139

Survey Number

Mammalian, aherentCell Type

Mouse, NIH/3T3 derived retroviral vector packaging cell linesSpeciesUsed

DNA: pBR322 derived plasmidscontaining retroviral vectors.


DMEM + 10% Fetal Bovine Serum + 1%Glutamine(GIBCO/BRL, Sigma)



5 min, with DNA at roomtemperature

Pre-pulseIncubation


Temperature

DMEM + 10% Fetal Bovine Serum + 1%Glutamine




0.1 to1 µg / µlDNA Concentration


10 µlVolume of DNA


0.4 cmCuvette Gap

0.2 kVVoltage


960 µFCapacitor



DMEM + 10% Fetal Bovine Serum + 1%Glutamine

Outgrowth Medium




50 to 200 transformants / µg DNAElectroporationEfficiency


Bruce Sullenger - Graduate StudentName of Submittor

Memorial Sloan-Kettering Cancer CenterMolecular Biology Dept.1275 York Avenue, Cost Center 6050New York, NY 10021

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


140

Survey Number


Mouse, NSO, myeloma cellsSpeciesUsed

DNA: immunoglobulin genes in pSVvector


RPMI + 10% Fetal Calf Serum(GIBCO/BRL, Sigma)






12 x 10 (6) / mlCell Density




20 µlVolume of DNA


0.2 cmCuvette Gap

0.250 kVVoltage


960 µFCapacitor




0 °C for 10 min. (ice)Outgrowth Temperature


mycophenolic acid Selection Methodor Assay Used

NoneElectroporationEfficiency

40 to 50 %Per Cent Survival

Rosaria OrlandiName of Submittor

Istituto Nazionale TumoriOncoldgia SperimentaleVia Venezian 1Milano, 20100 ITALY

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


141

Survey Number


Mouse, SP2/0, myeloma [Sp-2]SpeciesUsed

DNA, 14.15 kB circular plasmid whichcontains a part of pSV2neo.


10% Fetal Bovine Serum /RPMI 1640(GIBCO/BRL,Sigma)

Cell Growth Medium 5.4 X 10 (5) cells / mlGrowth Phase atHarvest

Phosphate Buffered Saline, 2 times

Wash Solution

on ice for 10 minutesPre-pulseIncubation



1.25 x 10 (7) cell / mlCell Density


1.12 µg / µlDNA Concentration

0.1 x TE buffer (1x=10 mM Tris, 1mMEDTA, pH 8.0)


18 µlVolume of DNA


0.4 cmCuvette Gap

0.22 kVVoltage


960 µFCapacitor



10% Fetal Bovine Serum / RPMI 1640Outgrowth Medium



800 µg / ml of G418Selection Methodor Assay Used

50 µg DNAElectroporationEfficiency


Hiroyuki SeimiyaName of Submittor

University of TokyoInstitute of Applied Microbiology1-1-1 Yayoi Bunkyo-ku, Tokyo, 113Tokyo, Japan

InstitutionAddress


• FEBS Lett. 244:301 (1989). Y. Kurosawa et. al. Convenientplasmid vectors for construction of chimeric mouse/humanantibodies.• Cancer Research 50: 3167 (1990). T. Tsuruo et. al.Mouse-Human Chimeric Antibody against the multidrugtransporter P-Glycoprotein

PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4,1.15g Na2HPO4


The Pulse

Before the Pulse

After the Pulse



Medium*


142

Survey Number


Mouse, SP-2, myeloma [Sp2/0-Ag14].SpeciesUsed

DNA: linearized DNA used for stabletransfections.







Temperature


5 x 10 (5) cells/pulse, stable transfectionCell Density






0.4 cmCuvette Gap

0.180 kVVoltage


960 µFCapacitor






G418 (stable transfections)Selection Methodor Assay Used





InstitutionAddress


Note: Stable transfections generally do not use carrier DNA. Also,the level of selective agent required to kill off non-transfectedcellsneeds to be established before transfection. The levelrequired should kill non-transfected cells in approximately 7 days.


The Pulse

Before the Pulse

After the Pulse



Medium*


143

Survey Number


Mouse, D10.G4.1,T-cell, helperSpeciesUsed

DNAMoleculesElectroporated

DMEM (10% fetal calf serum) + 20% con A supplement(GIBCO/BRL, Sigma)

Cell Growth Medium log phaseGrowth Phase atHarvest

Phosphate Buffered Saline without Ca++, Mg++Wash Solution

10 to 15 min on icePre-pulseIncubation

Room TemperatureElectroporation

Temperature

Ca++, Mg++ freeElectroporationMedium

10 (7) cells / 800 µlCell Density


10 µg in sterile water or TEDNA Concentration




0.4 cmCuvette Gap

0.3 kVVoltage


960 µFCapacitor



DMEM (10% serum) + 20% con A supplementOutgrowth Medium



HygromycinSelection Methodor Assay Used

not quantified (used CAT assay)ElectroporationEfficiency


Yang Park, PostdocName of Submittor

UMDNJ-RWJMSPathology675 Hoes LanePiscataway, NJ 08854

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


144

Survey Number


Monkey, Vero, kidney cellsSpeciesUsed

















0.4 cmCuvette Gap

0.25 kVVoltage


960 µFCapacitor






Transient (CAT, β-gal, immunohisto- chemistry).Selection Methodor Assay Used





InstitutionAddress



The Pulse

Before the Pulse

After the Pulse



Medium*


145

Survey Number


Mouse, X-63, myeloma [p3 X63 - Ag8.653]SpeciesUsed

DNA: linearized DNA used for stabletransfections.







Temperature


5 x 10 (5) cells/pulse, stable transfectionCell Density






0.4 cmCuvette Gap

0.180 kVVoltage


960 µFCapacitor






G418 (stable transfections)Selection Methodor Assay Used





InstitutionAddress


Note: Stable transfections generally do not use carrier DNA.Also, the level of selective agent required to kill offnon-transfected cellsneeds to be established before transfection.The level required should kill non-transfected cells inapproximately 7 days.PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4, 1.15g Na2HPO4


The Pulse

Before the Pulse

After the Pulse



Medium*


146

Survey Number


Mouse cells, unspecifiedSpeciesUsed

DNA: circularMoleculesElectroporated

DMEMCell Growth Medium Log phaseGrowth Phase atHarvest




Temperature


10 (8) cells /250 µlCell Density






0.4 cmCuvette Gap

0.40 kVVoltage


25 µFCapacitor







Not knownElectroporationEfficiency


S. MatsubaraName of Submittor

Kagoshima UniversityDept. of Medicine8-35-1 SakuragaokaKagoshima City, JAPAN

InstitutionAddress

Note: exponential values designated in parentheses.Electroporation effiicencies may be enhanced by use of theCapacitance Extender (provides longer time constants).


The Pulse

Before the Pulse

After the Pulse



Medium*


147

Survey Number


Rat, submandibular acini (secretory cells).SpeciesUsed

Fluorescent dyes (SBFI and MQAE);(3)H-inositol.


KRH + CaCell Growth Medium Not applicableGrowth Phase atHarvest

Cell growth mediumWash Solution

A variation of the growth medium,but high in potassium and low insodium and calcium.

Pre-pulseIncubation


Temperature

Same as pre-pulseElectroporationMedium

10 mg protein / mlCell Density

10 mg protein / mlVolume of Cells

1 mg protein / mlDNA Concentration

Same as Pre-Pulse buffer or KRH + CaDNA ResuspensionBuffer

Not assayedVolume of DNA


0.4 cmCuvette Gap



25 µFCapacitor

(Pulse Controller) Ω none.Resistor


Not applicableOutgrowth Medium






Sheryll Barker, Research TechnologistName of Submittor

Lovelace Medical FoundationResearch Dept.2425 Ridgerest Dr. S.E.Albuquerque, NM 87108

InstitutionAddress


**It is NOT RECOMMENDED to use high voltage with out thePulse Controller.


The Pulse

Before the Pulse

After the Pulse



Medium*


148

Survey Number


Rat, fibroblastsSpeciesUsed

DNA, retroviral vector, pN2, 9 kB,linearized at Hind IIII site.


DMEM, 10% Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium 70% confluentGrowth Phase atHarvest

NoneWash Solution

2.0 minPre-pulseIncubation

25 ° CElectroporationTemperature

DMEM, 10% Fetal Calf SerumElectroporationMedium



0.4 µg / µlDNA Concentration


5 µlVolume of DNA


0.4 cmCuvette Gap

0.25 kVVoltage


500 µFCapacitor

(Pulse Controller ) Ω none Resistor


DMEM,10% Fetal Calf SerumOutgrowth Medium



G418 resistance, 500 µl / ml Selection Methodor Assay Used

1.21 x 10 (3) transfectants / µg DNAElectroporationEfficiency

Not recordedPer Cent Survival

Marc D. Corina, Graduate StudentName of Submittor

Baylor College of MedicineCell BiologyOne Baylor PlazaHouston, Texas 77030

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


149

Survey Number


Rat, L-6, myoblastSpeciesUsed






4° C, 10 min. (optional: add 50 µlFCS if using HeBS aselectroporation media; 50 µlsalmon sperm DNA for transienttransfections).

Pre-pulseIncubation


Temperature









0.4 cmCuvette Gap

0.350 kVVoltage


960 µFCapacitor











InstitutionAddress


HBS: 10mM HEPES, pH 7.2, 150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


150

Survey Number


Rat, H4-11-E-C3, hepatomaSpeciesUsed







Pre-pulseIncubation


Temperature









0.4 cmCuvette Gap

0.240 kVVoltage


960 µFCapacitor











InstitutionAddress




The Pulse

Before the Pulse

After the Pulse



Medium*


151

Survey Number


Ovine (sheep), R.E., rumenSpeciesUsed







Pre-pulseIncubation


Temperature









0.4 cmCuvette Gap

0.270 kVVoltage


960 µFCapacitor











InstitutionAddress


HBS: 10mM HEPES, pH 7.2,150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


152

Survey Number


Ovine (sheep), CSL503, fetal lungSpeciesUsed







Pre-pulseIncubation


Temperature









0.4 cmCuvette Gap

0.380 kVVoltage


960 µFCapacitor











InstitutionAddress


HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


153

Survey Number


Rat, D202CC, hepatoma; Human, TCCSUP (epithelial-like) bladdercarcinoma

SpeciesUsed

DNA: linearized, (pMSG-derivative)MoleculesElectroporated

MEM, 10% Fetal Calf Serum,(+aminopterin, MPA, hypoxanthin,xanthine, thymidine)(GIBCO/BRL, Sigma)


Phosphate Buffered SucroseWash Solution

10 min. on ice, in PhosphateBuffered Sucrose

Pre-pulseIncubation


Temperature

Phosphate Buffered Sucrose(272 mM sucrose, 7 mM potassiumphosphate, pH 7.4, 1 mM MgCl2)


1 to 50 x 10 (5) cells / mlCell Density




5 to 20 µg /mlVolume of DNA


0.4 cmCuvette Gap



25 µFCapacitor



As aboveOutgrowth Medium


Stable transfectantsLength of Incubation

MPASelection Methodor Assay Used

1 to 10 x 10 (6) cellsElectroporationEfficiency


Mikael Widersten, Ph.D. StudentName of Submittor

BMCBiochemistry DeptBox 576S-75123 Uppsula, SWEDEN

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


154

Survey Number


Monkey, COS, kidney cells; Rat, N62 T cells; Mouse, mammaryepithelial cells

SpeciesUsed

DNA: linear & supercoiled: Rsv neo, Rsvgal, myc CAT, IRF CAT, fos CAT, hspCAT, etc.


Fischer's + 10% Horse Serum + 10% FetalCalf Serum (GIBCO/BRL)

Cell Growth Medium NoneGrowth Phase atHarvest

Wash Solution

0 to10 min, room temperature Pre-pulseIncubation


Temperature

Hepes buffered saline or Fischer's + 10%Horse Serum +10% Fetal Calf Serum


1 x 10 (7) cells / 0.4 mlCell Density

0.4 ml to 0.8 mlVolume of Cells

80 µg/0.3 ml Hepes BufferedSalineDNA Concentration


< 50 µlVolume of DNA


0.4 and 0.2 cmCuvette Gap

0.25, 0.35, 0.45 kVVoltage

625, 875, 1125kV/cmField Strength

125, 250, 960 µFCapacitor



Fischer's + 10% Horse Serum + 10% Fetal CalfSerum

Outgrowth Medium



G418,400 µg / mlSelection Methodor Assay Used

poorElectroporationEfficiency


Li-yuan Yu-Lee, Assistant ProfessorName of Submittor

Baylor College of MedicineMedicine Department1 Baylor PlazaHouston, Texas 77030

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES, pH 7.2, 150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


155

Survey Number

Mammalian, adherent, suspension, Cell Type

Monkey, Vero, kidney cells; Human, C-4I, cervical carcinoma cellsSpeciesUsed

DNA: circular, 9.2kB, expression vectorsMoleculesElectroporated

M199(GIBCO/BRL, Sigma)

Cell Growth Medium 3 x 10 (6) cells / mlGrowth Phase atHarvest

M199Wash Solution



Temperature

M199ElectroporationMedium


0.3 ml / pulseVolume of Cells

100 µg DNA / pulseDNA Concentration

distilled waterDNA ResuspensionBuffer

5 µlVolume of DNA


0.4cmCuvette Gap

0.250 kVVoltage


500 µFCapacitor



M199 - DMEM /F12,5% FBS,5% serumsupplement, 1% pen/ strep, 400 µg G418

Outgrowth Medium


24 hours recovery before selectionLength of Incubation


1.3 / µg DNAElectroporationEfficiency


Cherrilee SteeleName of Submittor

UTHSC-DBMicrobiology Dept.P.O. Box 20068Houston, Texas 77225

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


156

Survey Number


Human, 293, kidney cells; Hamster, CHO, ovary cellsSpeciesUsed

DNA: Plasmid DNA, 5 kB -12 kBMoleculesElectroporated

Hamms F-12 / DMEM (50:50) +10% FetalBovine Serum (GIBCO/BRL, Sigma)



Change to Hamms F-12 / DMEM(50:50) media 24 hours prior topulse.

Pre-pulseIncubation


Temperature

High glucose/DMEM (Best we tested)ElectroporationMedium




TEDNA ResuspensionBuffer

2 µlVolume of DNA


0.4 cmCuvette Gap

0.245 kVVoltage

0.098 kV / cmField Strength

960 µFCapacitor



Hams F-12 / DMEM (50:50) media +10% FetalBovine Serum

Outgrowth Medium


20 minLength of Incubation




George CachiaresName of Submittor

Genentech Inc.Molecular Biology460 Pt. San BrunoSan Francisco, CA 94080

InstitutionAddress


We are currently testing amplification procedures with linearizedplasmid [See: Barsou, M., DNA and Cell Biology v.9 (4): 293-300].PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4, 1.15g Na2HPO4HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


The Pulse

Before the Pulse

After the Pulse



Medium*


157

Survey Number


Human, B lymphomas: BJAB, P3HR-1, B95-8; Rat-1SpeciesUsed

DNA: ccc Bam Z (5 kB), EcoA cosmid >40 kB, SalA cosmid > 40 kB


RPMI-10 (GIBCO/BRL, Sigma)Cell Growth Medium LogGrowth Phase atHarvest

NoneWash Solution


4°CElectroporation

Temperature

RPMI-10 + 15% serumElectroporationMedium

5 x 10 (6) or 10 (7) per 350 µlCell Density


10 to 50 µg / cuvetteDNA Concentration

RPMI-10 or Phosphate Buffered Saline(PBS) or water


10 µlVolume of DNA

Gene Pulser® apparatus, CapacitanceInstruments Used

0.4 cmCuvette Gap



960 µFCapacitor





VariableLength of Incubation

Protein expression by immunoblot,immunofluorescence, generation ofrecombinants.


> 20%ElectroporationEfficiency

Variable %Per Cent Survival

Sankar Swaminathan, M.D.Name of Submittor

Harvard Medical SchoolBrigham & Women's HospitalDept of Infectious Diseases20 Shattuck St.Boston, MA 02115

InstitutionAddress

Note: exponential values designated in parentheses.PNAS88:1546-1550 (1991).PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4,1.15g Na2HPO4


The Pulse

Before the Pulse

After the Pulse



Medium*


158

Survey Number


Human, HeLa, cervical (C41); Human epithelial cells; Monkey, Vero,kidney.

SpeciesUsed

DNA: circular plasmid recombinants(pMAM (8.8 kB + insert),pATH (9.0 kB) pGem (4.0 kB) pMSG (8.8kB) pM6 (9.7 kB)


DMEM, MEM, M199; (10% Fetal BovineSerum + 1% antibiotic) (GIBCO/BRL,Sigma)

Cell Growth Medium Early logGrowth Phase atHarvest

Phosphate Buffered Saline (cold)Wash Solution

Ice, 10 min.Pre-pulseIncubation

ChilledElectroporation

Temperature

Same as growth mediaElectroporationMedium

3 x 10 (6) cells / ml Cell Density


10 to 100 µg / sampleDNA Concentration


1 µl to 50 µlVolume of DNA


0.4 cmCuvette Gap

0.250 kV Voltage


500 µFCapacitor



Same as growth mediaOutgrowth Medium

Brief ice, then 37°COutgrowth Temperature

16 to 48 hours prior to selectionLength of Incubation


1 x 10 (-3) to 10 (-9) depending on cell type andplasmids used.

ElectroporationEfficiency


Dr. Cherrilee SteeleName of Submittor

Univ. of Texas Health Sciences Center Microbiology / DentalBranchP.O. Box 20068 / John Freeman Ave.Houston, TX 77225

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


159

Survey Number


Mouse, NIH/3T3, embryo; Human T-cell line, (PEER) SpeciesUsed

DNA: pTS1-envlF, 11 kB, supercoiledMoleculesElectroporated

MDEM (GIBCO/BRL, Sigma)Cell Growth Medium Not givenGrowth Phase atHarvest

NoWash Solution

MDEMPre-pulseIncubation


MDEMElectroporationMedium

75%Cell Density



TE (10 mM Tris, 1 mM EDTA, pH 8.0)DNA ResuspensionBuffer

35 µlVolume of DNA


0.4 cmCuvette Gap

0.270 kVVoltage


960 µFCapacitor



MDEMOutgrowth Medium


10 min.Length of Incubation


Very goodElectroporationEfficiency


Dr. Yuejin Yu, Postdoctoral FellowName of Submittor

University of TexasM.D. Anderson Cancer CenterScience Park Research DivisionPark Road 1C, P.O. Box 389Smithville, Tx 78957

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


160

Survey Number


Mouse, 3T3, embryo; Human, fibroblast; primary cell lines;Monkey,Vero, kidney cells.

SpeciesUsed

DNA: plasmids and cosmids, 40 kb andsmaller.


DMEM + 10% Nu serum(GIBCO/BRL, Sigma, Flow Labs)

Cell Growth Medium Passaged 24 hours prior to pulse.Growth Phase atHarvest


minimumPre-pulseIncubation


Opti medium (BRL)ElectroporationMedium

10 (7) cells / pulseCell Density


10 to 20 µg / pulseDNA Concentration

20 µl TE (10 mM Tris, 1 mM EDTA, pH8.0) / pulse


20 µlVolume of DNA


0.4 cmCuvette Gap

0.220 kVVoltage


960 µFCapacitor



DMEM + 10% Nu serumOutgrowth Medium



b-gal expression: X-gal staining. Replication ofHerpes origin of replication between S plasmids


30 to 50% cells transfectedElectroporationEfficiency


Dr. Marie MasseName of Submittor

Stanford UniversityMicrobiology and ImmunologyStanford, CA 94305

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


161

Survey Number


Human, HepG2, hepatoma; Monkey, COS-7, kidney.SpeciesUsed

cDNA's : supercoiled and linear.MoleculesElectroporated

RPMI,10% Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium Per Bio-Rad protocolGrowth Phase atHarvest

Per Bio-Rad protocol (see Gene Pulser Instruction Manual)Wash Solution

Per Bio-Rad protocolPre-pulseIncubation

0 °C (ice)Electroporation

Temperature


10 (7) cells / ml Cell Density




1 µl to 10 µlVolume of DNA


0.2 cmCuvette Gap

0. 30 kVVoltage


960 µFCapacitor



RPMI /10% Fetal Calf SerumOutgrowth Medium



FACSSelection Methodor Assay Used

100 transfectants/ µg DNAElectroporationEfficiency


J.S. MaltorName of Submittor

Tulane Medical CenterPathology Dept.1430 Tulane AvenueNew Orleans, LA 70112

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


162

Survey Number


Human, squamous cell carcinoma lines, oral & cervical;Monkey,Vero, kidney cells.

SpeciesUsed

DNA: Recombinant pMAM neo vectors(8.8 to 9.2 kB) - confirm via expression(Northern blot) immunoblotting, PCR &Southern blot


M199, MEM, DMEM/F12(GIBCO/BRL, Sigma)

Cell Growth Medium Mid-log phaseGrowth Phase atHarvest

Ice-cold Phosphate Buffered Saline, pH 7.2Wash Solution

Ice, 10 min., in media with DNA.Pre-pulseIncubation


Growth mediaElectroporationMedium


3 x 10 (6) cells / pulseVolume of Cells





0.4 cmCuvette Gap

0.250 kVVoltage


500 µFCapacitor



Growth media, plus or minus G418Outgrowth Medium


at least 10 daysLength of Incubation


6 x 10 (-5) to 9 x 10 (-8); depends on cell line usedElectroporationEfficiency


C. SteeleName of Submittor

UTHSC - DBDept of MicrobiologyP.O. Box 20068Houston, TX 77225

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


163

Survey Number


Human, fibroblast; Human Hep3b2, hepatocyte; Mouse, L-cells.SpeciesUsed

DNA: varies, fibronectin β-gal, genomicDNA, CMUB, etc.


MEM + 10% Fetal Calf Serum(GIBCO/BRL, Sigma)


Phosphate Buffered Saline and TrypsinWash Solution

5 minutesPre-pulseIncubation


MEMElectroporationMedium

1 to 10 x 10 (6) cells / pulseCell Density


40 to100 µg DNA per pulseDNA Concentration





0.4 cmCuvette Gap

0.320 kVVoltage


500 µFCapacitor



MEMOutgrowth Medium


48 hours to 1 monthLength of Incubation

GPT and NeomycinSelection Methodor Assay Used



Ranee Taylor - StudentName of Submittor

Baylor College of MedicineMolecular Genetics1 Baylor PlazaHouston, Texas 77030

InstitutionAddress


The Pulse

Before the Pulse

After the Pulse



Medium*


164

Survey Number


HybridomasSpeciesUsed

DNA: Antibody genes with selectablemarkers.


DMEM + 15% Hourse Serum + L- glutamine

(GIBCO/BRL, Sigma)

Cell Growth Medium Mid- logGrowth Phase atHarvest

Cold Phosphate Buffered SalineWash Solution

10 minutes on ice.Pre-pulseIncubation

4°CElectroporationTemperature








0.4 cmCuvette Gap

0.160 kVVoltage


960 µFCapacitor



DMEM + 15% Fetal Calf Serum + Selector Outgrowth Medium


> 1 weekLength of Incubation

G418 or mycophenolic acidSelection Methodor Assay Used

0.5 to 1 transfectants / µgElectroporationEfficiency


Dr. Richard NearName of Submittor

Mass. General HopsitalCardiology Dept.Jackson 1422 CardiologyBoston, MA 02114

InstitutionAddress


Immunol ., Jan 15, 1991.


The Pulse

Before the Pulse

After the Pulse



Medium*


165

Survey Number


Hybrid, mouse/human, A9 fibroblast, hybrid containing humanchromosomes

SpeciesUsed

DNA: linearized 8.5 kB plasmidMoleculesElectroporated

DMEM + 10% Fetal Calf Serum +mycophenolic acid + xanthine

Cell Growth Medium 10 (7) cells / mlGrowth Phase atHarvest

HEPES Buffered Saline (HBS)Wash Solution

10 min. on ice, (0°C)Pre-pulseIncubation


Temperature

HEPES Buffered Saline (HBS)ElectroporationMedium

Approximately 10 (7) cells / mlCell Density






0.4 cmCuvette Gap

0. 250, 0.450 kVVoltage

0.625,1.125 kV/cmField Strength

125, 250, 500, 960 µFCapacitor






Hygromycin BSelection Methodor Assay Used


Not given Per Cent Survival

Keith A. RosenbachName of Submittor

UMDNJ New Jersey Medical SchoolMicrobiology & Molecular Genetics185 South Orange Ave.Newark, NJ 07103

InstitutionAddress




The Pulse

Before the Pulse

After the Pulse



Medium*


166

Survey Number


Mouse, BbSutA, hematopoietic;Human, K562, HEL, HL 60 (leukemia lines)

SpeciesUsed

DNA, pN2 (retroviral vector),pCDM8-CD34 (both linears andsupercoil).


B6SutA: McCoy's 5A + supplements(15% FCS + 10% WEHI-CM) human lines:RPMI 1640 + 10% calf serum(GIBCO/BRL, Sigma)


Dulbecco's PBS, no Ca++ or Mg++Wash Solution

5 to 10 min., room temperaturePre-pulseIncubation


Temperature

Dulbecco's PBS, no Ca++ or Mg++ElectroporationMedium

5 x 10 (6) to10 (7) / mlCell Density


100 to 1000 ng / µlDNA Concentration





0.4 cmCuvette Gap

0.3 kVVoltage


500 µFCapacitor


5 to10 msecTime Constant

B6: McCoy's 5A + supplements, Leukemia lines:RPMI 1640 + 10% calf serum

Outgrowth Medium



G418 (100 to 800 µg / ml); cell survival.Selection Methodor Assay Used

UnknownElectroporationEfficiency

UnknownPer Cent Survival

Mayumi YagiName of Submittor

Seattle Biomed Reseach Inst.4 Nickerson St.Seattle, WA 98109

InstitutionAddress


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167

Survey Number


Monkey, CV-1, kidney; Mouse, 3T3, embryo; Mouse, p3x63AG8,myeloma; Human: HeLa, epithelial carcinoma

SpeciesUsed

ProteinsMoleculesElectroporated

DMEM (GIBCO/BRL, Sigma)Cell Growth Medium 50 to 60% confluent cellsGrowth Phase atHarvest

(See notes)Wash Solution

5 minutesPre-pulseIncubation


(See notes)ElectroporationMedium


0.2 µlVolume of Cells





0.4 cmCuvette Gap



25 µFCapacitor



Pore-Resealing Buffer (see notes) for 10 min. ,37°C; follow with DMEM plus 10% Fetal Calf Serum.

Outgrowth Medium

37° COutgrowth Temperature





Marcel R. Michel, Ph.D.Name of Submittor

Institute for Medical MicrobiologyMolecular BiologyFriedbuehlstrasse 513010 Berne, Switzerland

InstitutionAddress

Note: exponential values designated in parentheses.Ref:M.R. Mitchell et. al. (1988) Experentia 44: 199-203.M.R. Michel et. al. (1990) J.Virol . 64: 5123-5131.Elgizoli, M. et. al. (1989) J. Virol . 63: 2921-2928.Electroporation Buffer: 20 mM PIPES, pH 7; 128 mMK-glutamate, 5mM ATP, 5 mM GTP, 10 µm Ca-Acetate, 2 mMMg-Acetate & amino acid concentration corresponding to that ofDMEM media.Pore-Resealing Buffer: 20 mM PIPES, pH 7.0, 128 mMK-glutamate,10 µm Ca-Acetate, 2 mM Mg-Acetate


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168

Survey Number


Human, K562, chronic myeloid leukemia; Hamster, CHO, ovary;Hybrid, rat / mouse, MEL cells.

SpeciesUsed

DNA: linearized constructsMoleculesElectroporated

RPMI 1640, 10% Fetal Bovine Serum(GIBCO/BRL, Sigma)

Cell Growth Medium 3 x 10 (5) to 5 x 10 (5) cells / ml (suspension)Growth Phase atHarvest

Phosphate Buffred SalineWash Solution

10 min. at room temp in Hepesbuffered saline with dextrose

Pre-pulseIncubation


Temperature

HEPES buffered saline with dextroseElectroporationMedium




RPMI 1640 +10% Fetal Bovine SerumDNA ResuspensionBuffer



0.4 cmCuvette Gap

0.2 kVVoltage


960 µFCapacitor



RPMI 1640+10% Fetal Bovine SerumOutgrowth Medium






No name or address givenName of Submittor

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl, 0.2g KCl, 0.2g KH2PO4,1.15g Na2HPO4HBS: 10mM HEPES,pH 7.2,150 mM NaCl, 5 mM CaCl2


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192

Survey Number


Human, K562, chronic myeloid leukemia; HeLa, epithelialcarcinoma; HEL cells, eythroleukemia.

SpeciesUsed

DNA: pGL-luciferase vector[Promega]containing b-globin promoter ;co-porated SV 40, b-gal.


RPMI + 5% Fetal Calf Serum + 5% DCS

Cell Growth Medium 2 to 5 x 10 (5) cells / mlGrowth Phase atHarvest

Phosphate Bufferd Saline + 5% DCSWash Solution

10 min., icePre-pulseIncubation

25°CElectroporationTemperature

Hepes Buffered SalineElectroporationMedium



50 µg per pulse DNA Concentration


50 µlVolume of DNA


0.4 cmCuvette Gap

0.300 kVVoltage


960 µFCapacitor



RPMI + 5% Fetal Calf Serum + 5% DCS

Outgrowth Medium



luciferease, β- galSelection Methodor Assay Used

Not given ElectroporationEfficiency


Dr. C. Anthony BlauName of Submittor

University of Washington- Med CenterDepartment of Medical GeneticsRoom BB510 HSB1959 Pacific Avenue, NESeattle , WA 98006

InstitutionAddress


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193

Survey Number


Mouse, C127, fibroblast, mammary tumorSpeciesUsed

DNA: Bovine papilloma virus E1 in pML2dMoleculesElectroporated

DMEM + 10 % Fetal Calf Serum(GIBCO/BRL, Sigma)

Cell Growth Medium logarithmicGrowth Phase atHarvest

PBS (Phoshate Buffered Saline) and Electroporation MediumWash Solution

10 minutes on ice in DMEM + 10 %Fetal Calf Serum + BES

Pre-pulseIncubation


Temperature

DMEM + 10 % Fetal Calf Serum + BESElectroporationMedium

1.5 to 2.0 x10 (6) cells/ pulseCell Density


10 to 20 µg / pulseDNA Concentration



25 µlVolume of DNA


0.4 cmCuvette Gap

0.21 kVVoltage


960 µFCapacitor



DMEM + 10% Fetal Calf SerumOutgrowth Medium


3 days /transient; 2-3 weeks/ stableLength of Incubation

G418 resistance for stable transformationSelection Methodor Assay Used

20-30%ElectroporationEfficiency

30-40%Per Cent Survival

Dr. Belyavski MichailName of Submittor

Texas A&M UniversityDepartment of Medical Microbiology and ImmunologyCollege Station, TX 77834

InstitutionAddress

Note: exponential values designated in parentheses.PBS: 1x = 8g NaCl,0.2g KCl,0.2g KH2PO4,1.15g Na2HPO4


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Medium*


194

Survey Number


Human, lymphocytes, primarySpeciesUsed

DNA:MoleculesElectroporated

RPMI-1640 + 10 % Fetal Bovine Serum(GIBCO/BRL, Sigma)





Temperature

RPMI-1640 + 10 % Fetal Bovine SerumElectroporationMedium

5 x 10 (6) cells/ mlCell Density

250 µl / pulseVolume of Cells





0.4 cmCuvette Gap

0.250 kVVoltage


960 µFCapacitor

(Pulse Controller) ΩResistor








Steve DewhurstName of Submittor

University of Rochester Medical CenterDept. of Microbiology601 Elmwood Ave.Box 672Rochester, NY 14642

InstitutionAddress


Used conditions as pulished by Chen, et al., Bio-Rad TechnicalBulletin No.1348.


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195

Survey Number

Mammalian, adherent and suspensionCell Type

Rat, PC12,adrenal pheochromocytoma; Rat brain; Simian(monkey) COS, kidney cells;

SpeciesUsed

DNA: 2 to 4 kB, supercoiledMoleculesElectroporated

DMEM(GIBCO/BRL, Sigma)

Cell Growth Medium Stationary growthGrowth Phase atHarvest

Dulbecco's PBSWash Solution



Dulbecco's PBSElectroporationMedium

5 x 10 (6) cells/ mlCell Density

400 to 800 µlVolume of Cells

20 to 200 µg / mlDNA Concentration

TE (10 mM Tris, 1 mmM EDTA, pH 8.0)DNA ResuspensionBuffer



0.4 cmCuvette Gap

0.250 kVVoltage


25 to 500 µFCapacitor


0.6 to 16 msecTime Constant



daysLength of Incubation

Binding asssays / transients;G418 selection / stable


variesElectroporationEfficiency

variesPer Cent Survival

Dr.Catherine BitlerName of Submittor

Molecular PharmacologistSRI InternationalNeuroscience Department333 RavenswoodMenlo Park, CA 94025

InstitutionAddress


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Medium*


196

Survey Number

MammalianCell Type

Human: HeLa, epithelial carcinomaSpeciesUsed

DNA: pRC/CMV- Human 5-HT(10 β) receptorconstruct, linearized.[See notes]


EMEM + 10 % Fetal Calf Serum,+ Non-essential amino acids+ Pen / Strep

Cell Growth Medium Pre-confluentGrowth Phase atHarvest

Phosphate Buffered SucroseWash Solution


about 4 °CElectroporationTemperature

Phosphate Buffer Sucrose(see Gene Pulser Instruction Manual )


5 x 10 (6) cells / 800 µlCell Density



Phosphate Buffered SucroseDNA ResuspensionBuffer


Gene Pulser® apparatus & Pulse ControllerInstruments Used

0.4 cmCuvette Gap

0.4 kVVoltage


25 µFCapacitor

(Pulse Controller) 200 ΩResistor


Post pulse: (in cuvette) 10 min. on ice, thenEMEM + 10 % FCS,+ non-essential a.a.+ Pen / Strep

Outgrowth Medium


plated, split next dayLength of Incubation

G418 selection (800 µg/ml) 24 hours post pulseSelection Methodor Assay Used

100 transformants / µgElectroporationEfficiency

Not knownPer Cent Survival

Dr. Mark W. HamblinName of Submittor

GRECC 182B, Seattle VAMC &Dept. of Psychiatry & Behavioral ScienceUniv.of Washington School of Medicine1660 S. Columbia WaySeattle, WA 98108

InstitutionAddress


DNA: 7.2 kB linear ds DNA construct including 5.6 kB of themammalian expression vector pRC/ CMV (Invitrogen) and 1.8 kBof human genomic DNA including the coding region of the5-HT10b (5 HT-1B) serotonin receptor, linearized with Bgl II.

Phosphate Buffered Sucrose : 272 mM sucrose,7 mM sodium phosphate, pH 7.4, 1 mM MgCl2.

Ref: Biophys.Biochem. Res. Comm .184:752-759.


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Medium*


197

Survey Number


Rat, CA77, medullary thyroid carcinoma cell lineSpeciesUsed

DNA: supercoiled plasmid; lucifereasereporter gene


DMEM/F12 (1:1) + 10 % Fetal BovineSerum

(GIBCO/BRL, Sigma)

Cell Growth Medium Active growth, about 50 to 70 % confluentGrowth Phase atHarvest

Dulbecco's Phosphate Buffered Saline (minus Ca++, Mg++)Wash Solution



Temperature

Dulbecco's Phosphate Buffered Saline(minus Ca++, Mg++)


4 to 5 x10 (6) cells / 800 µlCell Density



TE (10 mM Tris, 1 mM EDTA)DNA ResuspensionBuffer

5 to 50 µl (usually less than 10 µl)Volume of DNA


0.4 cmCuvette Gap

0.22 kVVoltage


960 µFCapacitor


about 12 msecTime Constant

DMEM/F12 (1:1) + 10 % Fetal Bovine SerumOutgrowth Medium


usually 24 hoursLength of Incubation

Luciferase assay, β-galactosidase assaySelection Methodor Assay Used

about 1 x 10 (4) / µg [20 to 40 % cells stainedblue, b-gal, transient assay]

ElectroporationEfficiency

50 to 80 %Per Cent Survival

Andrew RussoName of Submittor

University of IowaDepartment of Physiology5-632 BSBIowa City, IA 52242

InstitutionAddress

Note: exponential values designated in parentheses.Reference: Tverberg, L.A. and Russo, A.F. 1992. J. Biol.Chem .267(5):17567-17573.


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Life ScienceGroup

07-0541 0707 Sig 1106D035551 US/EG Rev A

Bio-RadLaboratories, Inc.

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