fall 2009/spring 2010 prep guide bio:219 biotechnology...
TRANSCRIPT
EILENE LYONS – REVISED 1/12/2010 1
FALL 2009/SPRING 2010
PREP GUIDE
BIO:219 BIOTECHNOLOGY I
TABLE OF CONTENTS Setup for first day of class.....................Page 2
Lab 1 Genotyping of Arabidopsis.................Page 3-6
Lab 2 DNA Concentration and Purity .......... .....Page 7-8
Lab 3 PAGE.......................................Page 9- 10
Lab 4 Purification of Eco RI(EDVOTEK Kit 302)....Page 11-12
Lab 5 Mammalian Cell Culture...................Page 13 - 14
Lab 6 Pop Bead Cloning (dry lab)....................Page 15
Lab 7 DNA Restriction for Cloning...................Page 16
Lab 8 Gene Clean....................................Page 17
Lab 9 Ligation.................................... Page 18
Lab 10 Transformation & Blue-White Selection ....Page 19-20
Lab 11 Plasmid DNA Isolation (Miniprep) ........... Page 21
Lab 12 Recombinant DNA Restriction and Mapping .....Page 22
Lab 13 Manual DNA Sequencing (EDVOTEK Kit 341)...Page 23-25
Lab 14 Gene Expression (IL-8 ELISA KIT; Qiagen RNA cleanup
kit)............ ........... ........... .......Page 26
*Items marked with an asterisk do not need to be place on the
cart, but should be available in the room or in the immediate prep
area.
BIOTECHNOLOGY I FIRST DAY SETUP
EILENE LYONS – REVISED 1/12/2010 3
FIRST DAY SETUP Clean lab coats – all sizes
Colored water for pipetter practice
MATERIALS for growing Arabidopsis – see Lab 1 prep sheet
FOR EACH TEAM – set up one bin for each team, please
automatic micropipetters - one each size personal microcentrifuge & both rotors
beaker labeled for used tips plastic dish for staining/holding gels
Kim wipes scissors
loading dye - 6x or 10x Sharpie Markers -2
mm ruler sterile tips -one box of each kind of
microcentrifuge tubes - 1.5 ml -sterile tube rack -15 ml
molecular grade water - 10 mL tube rack - 1.5 ml
IN THE CLASSROOM PERMANENTLY
Please make sure there is container for dirty glassware during each lab
Agarose – electrophoresis grade
Buffers: 50x TAE, 50x TBE, 1x TNE buffer, 10:1 TE buffer
Carboy of dH2O
Colored Owl or Fisher horizontal gel rigs with combs
Conical tubes, Fisher, 15 ml and 30 ml and racks
Coverage – one bottle per table
Cuvettes, cuvette racks
Electronic balances, weigh boats, spatulas and paint brush for clean up
Ethidium Bromide [10 mg/ml] stock (keep refrigerated – small refrigerator?)
Ethidium Bromide disposal station (for disposal of buffer and gels)
Glassware, including beakers, 250 ml flasks, graduated cylinders, Corning bottles
Heating block with holes for 1.5 ml tubes - 2 Kim wipes
Lab diapers
Labeling tape
Microcentrifuges – Eppendorf type – at least 2
Microcentrifuge tubes, 1.5 ml and racks
Microwave oven and hot gloves
Parafilm
Plastic wrap
Serological pipette pumps – manual and electric
Serological pipettes – all sizes
Sterile tips – all sizes
Transfer pipettes
Water bath and floating tube holders
Vortex mixers
Zip Lock bags for storing gels
ITEMS IN THE PREP AREA WHERE STUDENTS CAN ACCESS EASILY:
Ice buckets – leave in the ice room by ice machine
Power supplies
Rockers and shakers
UV spectrophotometers – 2
UV transilluminator, camera, film, face shields, spatulas
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010 4
LAB 1 GENOTYPING OF Arabidopsis TIMELINE
Day 1: Plant Arabidopsis seeds and prepare solutions
Day 2: DNA extraction and PCR
Day 3: Gel electrophoresis and analysis
MATERIALS for growing Arabidopsis Arabidopsis seeds (Carolina Biological Supply or [email protected] for seed ordering)
Each student needs about 25-50 clf-2 seed (from a heterozygous plant);
Modified MS agar plates, one per student
Growth chamber set on continuous light
Bleach for 30% bleach solution (students will prepare)
Sterile dH2O – one tube or bottle per group
Sterile 0.1% agarose solution (students will prepare)
Aluminum foil
MATERIALS for DNA Isolation and PCR
2 per group Ready-To-Go PCR Beads (PuReTaq Ready-To-Go™ PCR Beads from
http://www1.amershambiosciences.com 96 reactions #27-9559-01, NC9711585 from Fisher, will
last ≥ 1.5 years; cost $227.50)
Each pellet reconstituted to 25 μL contains 1.5 units Taq polymerase, 10 mM Tris-HCl
(pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 200 μM of each dNTP.
Primers can be ordered from http://invitrogen.com
0.4 uM minimum CLF (wildtype) primers/loading dye mix – 25 μL per Team
FORWARD 5’-TTAACCCGGACCCGCATTTGTTTCGG-3’
REVERSE 5’-AGAGAAGCTCAAACAAGCCATCGA-3’
0.4 uM minimum clf-2 (mutant) primers/loading dye mix – 25 μL per Team
FORWARD 5’-TTAACCCGGACCCGCATTTGTTTCGG-3’
Ds REVERSE 5’-GTCGGCGTGCGGCTGGCGGCG-3’
Edward’s Buffer, 2 ml per team (Students will prepare)
Cresol Red Loading Dye
Isopropanol, aliquoted – 2 ml per team
Pellet pestles – one per team
Dissecting microscopes – 1 per team
Thermal cycler
Hair dryer
TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) – 500 μl per team
MATERIALS for Electrophoresis
100 - 1000 bp ladder or pBR322 DNA-BstN I digest (inexpensive marker from NE Biolabs)
*Heating block or water bath set at 50°C
*50X TAE electrophoresis buffer (students will dilute to 1X working solution)
*D.C. power supplies – per 2 groups
*Practice loading dye
*[10 mg/ml] ethidium bromide stock solution
*Should be in room – do not place on cart
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010
5
5
RECIPES:
Modified MS agar:
4.3 g MS salts (half the amount used for bacteria culture)
900 ml distilled water
Use 1.0 M NaOH or KOH to adjust to pH 5.7
BTV of 1 Liter
Then add 8 g agar and autoclave for 20 minutes at 120°C. One liter makes 40-50 10 cm
diameter plates.
Cresol Red loading dye
1% Cresol Red Dye Stock – yield 50 ml
Add 500 mg to 50 ml of ddH2O in a 50 ml tube or bottle
Shake to dissolve
Store at room temperature
Cresol Red Loading Dye – yield 50 ml of working solution
Add 17 g of sucrose to 49 ml of ddH2O in a 50 ml tube or bottle
Shake to dissolve
Add 1 ml of 1% Cresol Red Dye
Shake tube to mix. Store at 4°C.
Edward’s Buffer – yield 50 ml (solid NaCl and concentrated stocks will be used)
Mix the ingredients in a 50 ml bottle (can be stored at room temperature, indefinitely).
32.5 ml ddH2O
10 ml of 1 M Tris pH 8
2.5 ml of 5 M NaCl
2.5 ml of 0.5 M EDTA
2.5 ml of 10% SDS
50x Concentrated TAE Electrophoresis Buffer (40 mM Tris-acetate, 2 mM EDTA)
Add the following to dH2O to give a final volume of 1 liter
242 g Tris base (Tris [hydroxymethyl] aminomethane)
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8)
Students will dilute to a 1X working solution
Primer/Loading Dye Mix
Final concentration of components:
0.25 picomoles/μL of each primer, 13.9% sucrose, and 0.0082% cresol red in Tris-low
EDTA (TLE) buffer (10 mM Tric-HCl, pH 8.0; 0.1 mM EDTA).
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010
6
6
The ideal concentration is between 0.1 and 0.5 M. Primers are sold as solid DNA by
some companies and diluted in water by other companies. A certificate of analysis
packed with the primers give the mass, number of moles and if diluted, the concentration
in either μM or μg/μl or both. It can be difficult to determine how to dilute and mix the
primers for use in PCR so that the concentration is between the optimum 0.1 and 0.5 μM.
Table 1 and the sample calculation, below, may help.
Table 1. Molar conversion for Primer Concentration* Primer length pmol/µg 20 pmol** 18-mer 168 119 ng
20-mer 152 132 ng
25-mer 121 165 ng
30-mer 101 198 ng
* From Qiagen News, Issue 5 1997
** 20 pmol of primer in a 100 µl PCR reaction gives a primer concentration of 0.2 µM
SAMPLE CALCULATION for DILUTION OF LIQUID PRIMER DNA Suppose a primer concentration is given as 40 μM and 0.32 μg/μl. You need to make up 300 μl of primer/loading dye mix. The protocol states that 22.5 μl of the primer/loading dye is used in the 30 μl reaction. The final concentration of each of the primers in the 30 μl reaction must be 0.1 – 0.5 μM. 1. The primer’s final concentration in the reaction must be 0.1 – 0.5 μM, so dilute the
primer to 0.5 μM and use this to set up the reaction. 2. Use C1 x V1 = C2 x V2 to calculate the volume of working solution to use.
C1 = 40 μM
V1 = X C2 = 0.5 μM V2 = 300 μl
(40 μM) X = (0.5 μM) (300 μl) X = 3.75 μl
Add 3.75 μl of the primer as supplied by the manufacturer to enough loading dye and the other reagents to give a total of 300 μl. When 22.5 ul of this mix is used in the reaction, the final concentration of this primer will be 0.375 μM, which is within the 0.1 – 0.5 μM final concentration limits.
(0.5 μM) (22.5 μl) = X (30 μl) X = 0.375 μM
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010
7
7
SAMPLE CALCULATION for DILUTION OF DRY PRIMER DNA Suppose a primer is 280 μg and 50 nmoles and you need to make up 300 μl of primer/loading dye mix that has a final concentration of 0.25 picomoles/μl.
1. Dilute the primer to make a 100 pmole/μl stock solution. Add 500 μl of dH20 to dissolve the DNA.
50 nmoles = 50,000 pmoles 50,000 pmoles/500 μl = 100 pmoles/μl
2. Dilute 1 μl in 9 μl dH20 to give a working solution of 10 pmoles/μl. 3. Use C1 x V1 = C2 x V2 to calculate the volume of working solution to use. C1 = 10 pmoles/μl
V1 = X C2 = 0.25 pmoles/μl V2 = 300 μl
(10 pmoles/μl) X = (0.25 pmoles/μl) (300 μl) X = 7.5 μl
4. Add 7.5 μl working primer solution to make the primer/loading dye mixture.
BIOTECHNOLOGY I LAB 2 DNA CONCENTRATION AND PURITY
EILENE LYONS – REVISED 1/12/2010 8
LAB 2 - DNA CONCENTRATION AND PURITY
ITEMS TO ORDER IMMEDIATELY Commercial Lambda DNA (0.3-0.5 μg/μL)
DNase
High Mass Molecular Weight Marker DNA
TIMELINE
This lab will take 1 laboratory period
MATERIALS for PART I. Prep
10X TNE Buffer (please check to make sure there is no contamination in the bottle;
buffer may need to be filtered)
MATERIALS for PART II. 0.8% Gel Casting *Powdered agarose
*50x Concentrated TAE Electrophoresis Buffer (Students will dilute to 1X)
*Horizontal gel electrophoresis boxes – one/team
*Electronic balance, weigh-boats and spatulas
*Microwave oven and hot gloves
*Electronic pipette pumps
*Thumb-operated pipette pumps
*Serological pipettes – 10 ml
*100 ml graduated cylinder - one/team
*250 ml flasks - one/team
*Dishpan in sink for dirty glassware
*Scissors – one/team
*Sharpie marking pens
MATERIALS for PARTS III & IV Electrophoresis and UV Spectrophotometry
Commercial Lambda DNA (0.3-0.5 μg/μL) Place this and other DNA on ice for lab
1/20 dilution uncut concentrated Lambda DNA to use as unknown DNA samples – 50 μL
in a 1.5 mL tube labeled as DNA A per team – do not write concentration on tubes
1/10 dilution uncut concentrated Lambda DNA mixed 1:1 with Bovine Serum Albumin
to use as DNA B – 50 μL in a 1.5 ml tube per team
Degraded DNA – 50 μL in a 1.5 mL tube labeled as DNA C per team (add DNase)
REPORT DNA SAMPLE CONCENTRATIONS TO THE INSTRUCTOR
Molecular weight DNA ladder – one 1.5 ml tube per team
*6x or 10x Gel Loading Solution – one 1.5 ml tube per team
*Practice Gel Loading Solution – one 1.5 ml tube per team
*50X TAE electrophoresis buffer (students will dilute to 1X)
*1x TNE Buffer – two 50 mL bottles
*2 Beckman UV Spectrophotometers and matching quartz cuvettes for each
*Kim wipes
*Ethidium bromide [10 mg/ml]
* Place on cart if not in the storage cabinets in the lab
BIOTECHNOLOGY I LAB 2 DNA CONCENTRATION AND PURITY
EILENE LYONS – REVISED 1/12/2010
9
9
RECIPES for Lab 2
10x TNE Buffer stock solution:
100 mM Tris base (Tris [hydroxymethyl] aminomethane)
10 mM EDTA
2.0 M NaCl
Adjust pH to 7.4 using concentrated HCl and a pH meter. Dilute to 1x working
concentration using dH2O. Filter sterilize to remove all precipitate and contaminants.
50x Concentrated TAE Electrophoresis Buffer (40 mM Tris-acetate, 2 mM EDTA)
Add the following to dH2O to give a final volume of 1 liter
242 g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8)
BIOTECHNOLOGY I LAB 3 PAGE
EILENE LYONS – REVISED 1/12/2010 10
LAB 3 POLYACRYLAMIDE GEL ELECTROPHORESIS OF PROTEINS
TIMELINE
DAY 1: Prep for the lab, extract plant proteins, run SDS PAGE, stain and destain
DAY 2: Graph results using MS Excel and analyze
MATERIALS:
Per class
Bio-Safe Coomassie Blue stain (destains with dH2O)
*Practice gel loading solution
10X Tris-glycine-SDS buffer stock
*Vortex mixers
*Heating block set at 95°C
*1000 mL graduated cylinder
*dH2O
*1 L Corning orange capped bottles
*Plastic wrap
White light box
*Polaroid camera and film
*A supply of transfer pipettes
*Labeling tape
*1.5 ml microfuge tubes
Per every 2 teams
Flat metal spatula for separating gels
Dual vertical mini gel rig with clamps – please put on cart
Power supply – please put on cart
Per team
Kaleidoscope prestained protein molecular weight markers – aliquot 25 μl per team (Bio-
Rad # 161-0324) NOTE: heat briefly at 37°C to dissolve any precipitated SDS before aliquoting.
Flowering plants, one/team
2x Protein loading dye/buffer (≥ 100 ul)
Scalpel with sharp blade
Precast 15% polyacrylamide gel for SDS PAGE
8 disposable pellet pestles
2 ml 1X Laemmli buffer
*Should be in room – do not place on cart
BIOTECHNOLOGY I LAB 3 PAGE
EILENE LYONS – REVISED 1/12/2010
11
11
RECIPES
10x Tris-Glycine-SDS Buffer (500 ml should be allowed for each double gel rig; 1x =
25 mM Tris; 192 mM glycine, 0.1 % SDS )
3.04 g Tris base
14.41 g glycine
1.0 g SDS
dH2O to give 100 ml final volume
Dilute 100ml in 900ml dH2O for 1x working concentration
10 x Laemmli Buffer
0.25 M Tris,
1.92 M Glycine
1 % SDS in aqueous solution
Dilute to 1x working concentration
2x Protein loading dye (10 ml)
1.2 ml 1M Tris HCl pH 8**
4 ml 10% SDS
2 ml 100% glycerol
1 mg bromophenol blue
0.1 ml -mercaptoethanol
2.7 ml dH2O to give 10 ml final volume
**Adjust to pH 8 with HCl for prepoured graduated gels; if using discontinuous self poured
gels, adjust pH to 6.8.
BIOTECHNOLOGY I LAB 4 PURIFICATION OF ECO RI BY ION EXCHANGE
EILENE LYONS – REVISED 1/12/2010 12
LAB 4
PURIFICATION OF ECOR I by ION EXCHANGE CHROMATOGRAPHY
Edvotek Kit 302 is used for this lab
TIMELINE Day 1 – Prep, collect column fractions, cast agarose gels
Day 2 – First Assay
Day 3 – Second Assay
MATERIALS & EQUIPMENT – DAY 1 Chromatography columns, one per group
Ring stand with clamps, one per group
Ten 13 x 100 mm test tubes per group
(C) 10x Equilibration Buffer
Prep personnel should prepare the following
(B) DEAE-Cellulose (Please prepare 30 minutes before class per instructions, below)
(A) E. coli RY extract, lyophilized – must be re-hydrated per directions below
Equilibration Buffer working solution – DO NOT PREPARE UNTIL 10X HAS BEEN
USED TO HYDRATE THE DEAE-CELLULOSE
KCl buffers [0.1M], [0.2M], [0.5M] – each group will need 6 mL of each
Students will aliquot the following from the kit – Please place on the cart
(F) Eco RI Reaction Buffer
(H) Lambda DNA – 0.2 μg/μl concentration
(I) Lambda/Eco RI Marker
(J) Eco RI Dilution Buffer
Check the room/prep shelves for availability of the following: *50x TAE Electrophoresis Buffer (also in kit)
*15 ml conical centrifuge tubes
*Agarose powder
*Gel electrophoresis units, one per group
*5 ml serological pipette and pump, one per group
*Microwave oven & hot gloves
*Balances, spatula, weigh boats, cleaning brush
*Ice buckets
*1 Liter Corning bottles with lids
*[10 mg/ml] Ethidium bromide solution
*Should be in room or students can get – do not place on cart
RECIPES AND DIRECTIONS FOR DAY 1 PREP
DEAE-Cellulose Matrix
Add 35 ml of 10x equilibration buffer (C) to the bottle of DEAE-Cellulose (B). Cap
tightly and place on a rocker or orbital shaker to hydrate for at least 30 minutes. Aliquot 6
ml for each of the six groups. (The Instructor may have to do this)
(Recipes continued on the next page)
BIOTECHNOLOGY I LAB 4 PURIFICATION OF ECO RI BY ION EXCHANGE
EILENE LYONS – REVISED 1/12/2010 13
DAY 1 PREP (CONT.)
Equilibration Buffer Working Solution
Mix the following and stir thoroughly:
350 ml dH2O
50 ml 10x Equilibration buffer (C)
100 ml 50% glycerol (D)
KCl Buffers
0.1 M KCl: use 0.75 g KCl and BTV of 100 ml with Equilibration Buffer working
solution
0.2 M KCl: use 1.5 g KCl and BTV of 100 ml with Equilibration Buffer working
solution
0.5 M KCl: use 3.75 g KCl and BTV of 100 ml with Equilibration Buffer working
solution
E. coli Cell Extract containing Eco RI restriction enzyme
1. Re-hydrate the sample by adding 0.5 ml of ddH2O to tube component A and let sit for
5 minutes.
2. Mix by vortexing on high speed and transfer the entire contents to a 50 ml conical
tube. Rinse tube A six times – each time with 1 ml of Equilibration Buffer working
solution and add the rinse material to the 50 ml conical tube. Mix the contents well.
3. Label a tube for each of 6 lab groups as “Cell Extract” and add 1 ml of the re-
hydrated extract to each tube. Store this extract on ice.
MATERIALS AND EQUIPMENT - DAY 2 and DAY 3 *37°C Waterbath and floating tube holders (Each group will have 9 reaction tubes)
*65°C heating block with holes for 1.5 ml tubes (Each group will have 9 reaction tubes)
Lambda DNA diluted to 4 ng/uL; aliquot 25 ul for each group (this is needed for the gel
for the second assay)
[10 mg/ml] Ethidium bromide stock
*2 UV Spectrophotometers
*UV or Matched Quartz Cuvettes
*Dishes for transporting gels
*Plastic wrap
E-gels and E-gel rigs – one per team
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I MAMMALIAN CELL CULTURE
EILENE LYONS – REVISED 1/12/2010 14
LAB 5 – MAMMALIAN CELL CULTURE Here are a couple important things to prep before bringing up cells:
1. Sterilize the carbon dioxide incubator (for the older CO2 incubator, clean with 70% EtOH and
wipe with Beta-iodine, including shelves. If necessary, add water in the bottom after cleaning
and add Fisher Clear Bath.)
2. PLEASE CLEAN THE WATERBATH that will be used to warm culture media - wash with
antibacterial soap or detergent and add Clear Bath.
3. Autoclave media bottles- 100ml bottles. This is important to ensure no contamination. Even if
they have been already autoclaved once, couple days before autoclave again to ensure sterile,
remind whoever is doing this not to touch near the neck or bottle top. Store these bottles away
from any bacteria plates or incubators.
4. Pay special attention not the leave any supplies that will be used for the lab near bacterial
plates, incubators, or anything of the sort.
5. If instructor is NOT having students bring up cells, bring them up and passed at least once, to
make sure they are healthy. Do this the week prior to the lab. ATCC instructions come with
the cells.
TIMELINE: This lab will be continued through the remainder of the semester
MATERIALS
TO ORDER:
Cell scrapers (check with instructor to determine if she will use these or Trypsin-EDTA)
Please make sure all HEPA filters on the incubator and the safety cabinets are fresh
CO2 gas
NIH 3T3 Cells from ATCC (mouse embyonic cell line), item CRL-1658 ($203 Betsy’s price)
Then to grow the cells: (all reagents from Sigma)
D5796 media 6 x 500 mL $87.10
N4637 serum 500 mL $48.40
T3924 trypsin 2 x 100 mL $8.70 each
H6648 salt 6 x 500 mL $73.30 (this is Hank's balanced salt solution, if you want to
just have PBS made up, that is fine too)
PREPARE:
100X penicillin/streptomycin (check with instructor before ordering – may not be used)
Trypan Blue – aliquot 50 µL per 1.5 ml microcentrifuge tube per team
0.05% Trypsin-EDTA
Phenol red (pH 6.8-yellow – 8.2-red indicator)
Sterile Phosphate Buffered Saline (PBS), 1M OR Hank’s balanced salt solution
Sterilize CO2 Incubator
For Bringing up Cells prior to student lab
Flasks- T-25, T-75
Media: 100 ml of 10% FBS DMEM (+ Antibiotics, if instructor wants this) – students may
prepare media themselves
*Serological Pipettes
*15 ml Conical Centrifuge Tubes (Sterile for Serum and Antibiotics)
*sterile 50 ml tubes and Autoclaved or Sterile 100 ml Media bottles (Glass is better)
BIOTECHNOLOGY I MAMMALIAN CELL CULTURE
EILENE LYONS – REVISED 1/12/2010 15
Per Class (Amounts depend on # of teams):
*Confluent cell cultures in T-25 flasks, started 1 week prior
*DMEM or other media (in cold room)
10% Fetal Bovine Serum (FBS)
Sterile Phosphate Buffered Saline (PBS), 1M or Hank’s – divided into two bottles
Autoclaved or Sterile 100 ml Media bottles (Glass is better)
*5 ml, 10 ml, 25 ml serological pipettes
Cell scrapers (if using)
Flasks - T-25
Spray bottle of 70% EtOH
*Parafilm
*15ml tubes and holders (racks)
Hemocytometers – one per team
Cell counters one per team
Trypan Blue –50 µL per team
1 100ml Media Bottle per team
*sterile 50 ml tubes
*Light microscopes
Inverted light microscope – 1 with camera and computer and another 1 or 2 without
Water bath (clean the one in the lab and replace with fresh water)
*Labeling tape
To be left in the BSCs in the prep area and micro lab
10% bleach solution
container for used pipettes
two electric pipette pumps
beaker for old media sterilization
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I LAB 6 POP BEAD CLONING
EILENE LYONS – REVISED 1/12/2010 16
LAB 6
POP BEAD CLONING
DNA model
Pop bead cloning kit – please check to make sure each bag contains the correct number of
beads.
MATERIALS In your bead kit, you should have one linear set of beads in the following order:
Hole end, 13 White, 2 pink, 1 white, 1 pink, 1 yellow, 2 white, 2 red, 2 yellow, 4 red, 2
white, 2 yellow, 2 white, 1 pink, 1 yellow, 7 white, knob end.
There should also be one circular set of beads in the following order, starting after the
twisted white bead:
Hole end, 5 orange, 2 pink, 1 orange, 3 blue, 1 pink, 1 yellow, 3 blue, 1 orange, 2 yellow,
2 orange, 1 twisted white bead- knob end.
BIOTECHNOLOGY I LAB 7- DNA RESTRICTION FOR CLONING
EILENE LYONS – REVISED 1/12/2010 17
LAB 7 DNA RESTRICTION FOR CLONING
NOTE: Each team will set up one set of two digestions (pUC and pAMY).
TIMELINE: Students will set up digestions and run the results on an E-gel to confirm,
all in one class period
MATERIALS - DNA Digestion
Stock tube of pUC 18 DNA approx. [0.5 ug/ul]; students will dilute per instructor’s
directions
*Eco RI endonuclease
*10x buffer for Eco RI, 50 µL/team
*molecular grade dH2O
*Water bath set at 37 C
*automatic micropipetters and tips
*used tip container
*Kim wipes
*1.5 ml microcentrifuge tubes
*microcentrifuge tube rack
*Sharpie marking pen
Floating microfuge tube racks for water bath
MATERIALS - Gel Electrophoresis
E-gels and rigs
Uncut pUC 18 DNA as control on gel
Molecular Weight Marker DNA (with 10 L concentration labeled)
*dH2O
*automatic micropipetters and tips
*Microfuge tube rack
*UV Transilluminator
*UV Camera and film
*UV eye protection and face shields
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I LAB 8 GENE CLEAN
EILENE LYONS – REVISED 1/12/2010 18
LAB 8
Gene Clean
TIME LINE:
Day 1: Cast agarose gels for gene clean (on day of last lab.)
Day 2: Run the gel and extract the DNA fragments and gene clean; run the gene clean
product on an E-Gel to verify and quantify Mrs. Lyons will start the gel 2 hours before
class starts.
MATERIALS
55 C & 65°C water baths (with floating tube racks) – please make sure there are two in the lab
Please make sure 2 or more Eppendorf microcentrifuges are in the lab.
Sigma GenElute Gel Extraction Kit – Please dilute enough of each solution so that each
student has a 1.5 ml tube of each for his/her own gene clean.
*1X TAE buffer
Sterile swabs
*Student DNA digestions
Molecular Weight Marker DNA
*Kim wipes
*D.C. power supply
*UV Transilluminator
*UV Camera and film
*UV safety goggles and face shields
*[10 mg/ml] Ethidium Bromide stock
*6x or 10x gel loading dye
single-edge razor blades or scalpels
An E-gel and rig for gene clean
verification
*Molecular grade water
*disposal for Ethidium Bromide waste
*Should be in room – do not place on cart
BIOTECHNOLOGY 1 LIGATION
EILENE LYONS – REVISED 1/12/2010 19
LAB 9
LIGATION
MATERIALS
per class
*T4 ligase (check to make sure we have and let instructor know where it is)
*Gene cleaned insert DNA
*Digested plasmid DNA
Cooling block set at 16 C
*Vortex mixers – one per each end of lab bench
*Labeling tape
per Team
*Molecular grade or qualified water
*1.5 microcentrifuge tubes
10X Ligase buffer
ATP - 20 mM
*ice and ice bucket
*personal microcentrifuge
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I TRANSFORMATION
EILENE LYONS – REVISED 1/12/2010 20
LAB 10
TRANSFORMATION Order 95 mm x 15 mm plates – we will try larger plates this year.
Note: Each team will plate transformed culture onto 3 large plates. Two teams will also plate
negative control transformations onto 3 plates each and three teams will plate 3 positive control
plates each. Streak plates will also be made the day after and colonies picked off of that plate for
the miniprep, which means we need one more plate per team. So, 15 large plates are required
for the controls, plus 4 large plates per team, but have some extra, also. Make them all L-agar
+ Amp50
+ IPTG + X-gal.
MATERIALS For TRANSFORMATION
Per Class A tube of plasmid DNA from a previous semester for a control transformation (and back-up)
L-agar + Amp50
+ IPTG + X-gal plates – see above (room temp.)
JM109 Competent cells – 1 tube per team, and one for each of 5 controls
Recovery broth (SOC)
*Labeling tape to attach tubes to floor of shaker-incubator
*42 C heating block for transformation (check instructions with competent cells)
Shaker-incubator set at 37 C
Incubator set at 37 C for plates
Per team
*Qualified water
*1.5 ml microcentrifuge tubes – sterile – please check supply in room!
*ice and ice buckets with the following tubes to be assembled by the teams:
1 tube of supercoiled control DNA (supplied with competent cells)
-Mercaptoethanol (if supplied with competent cells – usually not with JM109)
Ligation reaction from previous lab
Qualified water
Sterile plastic spreaders for plating cells
*Personal microcentrifuge
MATERIALS FOR LIQUID CULTURE INOCULATION and GROWTH
Per class
Sterile toothpicks or sterile inoculating loops
Two bottles of 100 ml sterile L-broth media
Two 1.5 ml tubes of Ampicillin stock [10 mg/ml], thawed
10 ml (16 mm) sterile test tubes with loose fitting caps (two per student)
One test tube rack must be wired into the shaker incubator at an angle, so that the liquid cultures
shake at a slant and receive aeration, which is required for growth.
*Test tube racks – one more than the number of groups in the class
*Sterile 5 ml serological pipettes and pipette pumps
Shaker-incubator set at 37 C
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I TRANSFORMATION
EILENE LYONS – REVISED 1/12/2010 21
SOLUTIONS AND MEDIA for TRANSFORMATION
L agar-Amp50 -X-gal - IPTG plates (1 liter) Make plates within one week of the lab.
Make enough L agar for 40-45 mls per large Petri plate. Mix 10 g Bacto-tryptone (Difco
#0123-17-3), 5 g Bacto-yeast extract (Difco #0127-17-9), 10 g NaCl, 15 g Bacto agar (Difco #
0140-01), and cold ddH2O to make 1 liter. Use containers at least twice as large as the volume
of agar, or it may boil over in the autoclave. It is not necessary to boil the solution, as the
autoclaving will dissolve and mix the agar with the ddH2O. Autoclave 15 - 20 minutes at 15
p.s.i.
After agar has cooled to 50 C (agar is cool enough when you can hold your hand on the flask
with no discomfort), add sterile antibiotic (50 g/ml final concentration for ampicillin; use 10 ml
of 10 mg/ml sterile chloramphenicol). Final concentration of X-gal should be > 40 g/ml (use
3 ml 20 mg/ml sterile stock). Final concentration of IPTG should be > 120 g/ml (use 10 ml 20
mg/ml sterile stock). Stock solution instructions are below. (This is revised per Ginny after 04-
05.)
Pour into petri dishes. Let plates cool to room temperature. You may want to let them sit out
overnight at room temperature to get rid of the condensation on the lids. Sleeve plates, upside
down, in the plastic bag they came in, and store at 4 C. Bring to room temperature before using.
X-gal 20 mg/ml stock.
Dissolve 200 mg in 10 ml dimethyl formamide in a glass, not plastic, test tube with lid. Do not
filter sterilize. Store in dark (wrap aluminum foil around tube) at -20 C. Dimethyl formamide is
sterile.
IPTG 20 mg/ml stock
Dissolve 200 mg in 10 ml dH2O. Filter sterilize into sterile 15 ml centrifuge tube using a 0.22
micron filter attached to a 10 cc syringe.
Antibiotic stock solution- [10mg/ml]
Dissolve 100 mg in 10 ml ddH2O (or 95% ethanol for chloramphenicol). Filter sterilize into 10
sterile 1.5 ml microcentrifuge tubes using a 0.22 micron filter attached to a 10 cc syringe.
L-Broth (1 liter)
Use 10 g Bacto-tryptone (Difco #0123-17-3), 5 g Bacto-yeast extract (Difco #0127-17-9), 10 g
NaCl, and cold distilled ddH2O to make 1 liter. Aliquot to bottles. Autoclave 15 - 20 minutes at
15 p.s.i. After cooling, add sterile ampicillin (final concentration = 50 g/ml), if required.
SOC Broth
Buy pre-made. Contains the following:
2% Tryptone
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM glucose
BIOTECHNOLOGY I MINIPREP
EILENE LYONS – REVISED 1/12/2010 22
LAB 11
PLASMID DNA MINIPREP
MATERIALS
Miniprep
*Tubes of E. coli cultures grown overnight in shaking incubator
*Ice in ice bucket
*Kim wipes
*2 Eppendorf microcentrifuges we will not be using the personal microcentrifuges
*microfuge tube racks
*Sharpie marking pens
Promega Wizard Miniprep DNA isolation kit components - Please aliquot enough of each
solution so that every student can do two minipreps
Mini spin columns
3 mL syringes and plungers
*Molecular grade water
*container to dispose of bacterial waste
orange biohazard waste bag placed in the lab
*Coverage for cleaning bench tops
*10% bleach solution for disinfecting waste before disposal
Gel Electrophoresis
*plasmid DNA for + control on gel (instructor will get this)
*molecular grade water
E-Gels and rigs – one per every 2 teams (instructor should have E-gels)
Molecular Weight marker DNA
*Kim wipes
*UV Transilluminator
*UV Camera and film
*UV safety goggles and face shields
* carboy of dH2O
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I PLASMID MAPPING
Eilene Lyons Revised 1/12/10 23
LAB 12
RESTRICTION MAPPING TIMELINE
Day 1: Set up digestions of the recombinant plasmids that were constructed and isolated
in the previous labs; cast 0.7% agarose gels
Day 2: Run digestions on the gel, analyze results and construct the plasmid map(s).
MATERIALS:
PLASMID DNA RESTRICTION – DAY 1
per class
*Water bath set at 37 C with floatees
*Ice buckets with ice
*Thawed recombinant plasmid DNA
*Molecular grade water
*Nalgene ice tray for enzymes
*Eco RI endonuclease
*Xba I endonuclease
*Hind III endonuclease
*Restriction enzyme reaction buffers H,
B, C, D and E
*Universal buffer for double digestion
*1.5 ml microcentrifuge tubes
*microcentrifuge tube racks
*personal microcentrifuges
*Sharpie permanent markers
*TE buffer (to be used for dilution of
uncut plasmid DNA)
GEL CASTING – DAY 1
Per class
*balance, spatula and weigh boats
*microwave oven & hot gloves
*250 ml flask per group
* carboy of dH2O
*agarose
*horizontal gel electrophoresis rigs
GEL ELECTROPHORESIS OF DIGESTED PLASMID DNA – DAY 2
Molecular weight marker DNA (NOT High mass ladder – 0.5 and 0.1 bp markers are required)
*metric rulers
PUC18 vector DNA
*automatic micropipetters and tips
*used tip containers
*Kim wipes
*microfuge tube racks
*Sharpie marking pens
D.C. power supply – one per 2 groups
*UV Transilluminator
*UV Camera and film
*UV safety goggles and face shields
*50x TAE electrophoresis buffer
(students can dilute)
*10x gel loading solution
*Should be in room or students can get – do not place on cart
BIOTECHNOLOGY I DNA SEQUENCING
Eilene Lyons Revised 1/12/10 24
LAB 13
AUTOMATED DNA SEQUENCING MATERIALS
Heating block for 1.5 mL tubes, set to 95 C.
Molecular Grade (m.g.) sterile dH2O
m.g. 95% (v/v) ethanol/dH2O
m.g. 70% (v/v) ethanol/dH2O
3M Sodium Acetate pH5.2 – Sigma Cat. # S 7899
100 mM Na2EDTA pH8.0, prepared from 0.5 M Na2EDTA – Sigma Cat. # E 7889)
0.5 mL sterile microfuge tubes
Automatic micropipetters and tips
Thermal cycler with hot bonnet
Personal microcentrifuges with 0.5 tube rotor - one per group
Stop Solution (recipe below)
Materials from Beckman Coulter:
CEQ Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (No. 608120) which
contains:
Quick Start Mix
dNTPs
ddNTPs
Tris-HCl, MgCl2, reaction buffer – pH 8.9
Thermo Sequencase DNA Polymerase
Pyrophosphatase
pUC18 Control Template (0.25 g/ L)
M13 –47 Sequencing Primer (1.6 pmol/ L or 1.6 M)
Glycogen (20 mg/mL)
Mineral Oil
Sample Loading Solution (SLS)
Store the CEQ Cycle Sequencing kit at -20 C (frosty freezer, not frost free)
Recipes
Stop Solution (1.5 M NaOAc + 50 mM EDTA made fresh daily)
Mix equal volumes of 3M NaOAc and 100 mM EDTA
BIOTECHNOLOGY I ELISA
Eilene Lyons Revised 1/12/10 25
LAB 14
ELISA
MATERIALS
Per class:
Water bath set at 37 C
EDVOTEK Kit #271
HIV antigens (simulated – component A)
Positive control (1 antibodies – component B)
Donor 1 serum (simulated – component C)
Donor 2 serum (simulated – component D)
Anti-IgG-peroxidase conjugate (2 antibody – component E)
Hydrogen peroxide (component F)
Amiosalicylic acid (peroxide co-substrate – component G)
Phosphate buffered saline concentrate (component H)
Microtiter plates
Microcentrifuge tubes – 1.5 ml
Two 50 ml plastic tubes for mixing 2 antibody and substrate Scissors
Sharpie markers
Colored pencils
Tubes or bottles to hold 25 ml – one per student up to 10 per class (for PBS)
Beakers for waste solutions – one per two students
1 Corning bottle to hold 270 ml
Graduated cylinder to measure 270 ml
Electric serological pipette pumps – one per two students
1 ml serological pipettes – one per student
1 25 ml serological pipette
Bottles of dH2O – one per two students
BIOTECHNOLOGY I GENE EXPRESSION
Eilene Lyons Revised 1/12/10 26
LAB 15 GENE EXPRESSION IN MAMMALIAN CELLS
MATERIALS
Antibiotic media
10 ml Penicillin-Streptomycin
1 Liter F12 K media
110 mL Fetal Bovine Serum (FBS)
Filter sterilize
Trypsin-EDTA (1x)
1x Phosphate Buffered Saline (PBS without MgCl, without CaCl)
BioSafety I Epithelial cell culture at ≥ 80% confluence
24 well cell culture cluster plates with flat bottom and lid (Corning Inc. Costar #3524)
Sterile P-1000 pipet and tips (place in BSC during UV light sterilization)
Sterile 15 ml Corning orange capped tubes
BIOTECHNOLOGY I DNA TYPING
Eilene Lyons Revised 1/12/10 27
LAB 16
HUMAN DNA TYPING USING PCR
TIMELINE
DAY 1: Cheek cell DNA will first be isolated. The PCR reactions will be set up and
amplified in a thermal cycler. (1.5% agarose gel may be cast, if time.)
DAY 2: A 1.5% agarose gel will be cast. PCR reactions will be run, stained,
photographed and analyzed.
MATERIALS FOR PCR (DAY 1):
D1S80 primer mix (label as primer
mix)
Tris buffer (label as Tris buffer)
Chelating agent (label as resin)
10x PBS (label as 10x PBS)
200 base pair marker DNA (label as 200
bp marker)
PCR Tubes with beads that contain:
dNTP nucleotide mixture
Taq DNA polymerase buffer
Taq DNA polymerase
MgCl2
20 mL graduated cylinder
200 mL graduated cylinder (to measure
150 mL)
250 mL beaker
1.5 mL microcentrifuge tubes
1.5 mL conical tubes with screw caps
15 mL conical tubes with screw caps
racks for 15 mL tubes
sterile swabs- NOT BUCCAL SWABS!
Thermal cycler
Microcentrifuges
Vortex mixers
Centrifuge and rotor that accommodates
15 mL conical tubes
Automatic micropipetters and tips
Tips for PCR with aerosol barrier
Beaker for used tips
Heating block at 100 C (1.5 mL tubes)
dH2O
10 mL serological pipettes
electric pipet pump
250 mL flasks or beakers
ice buckets and ice
MATERIALS FOR GEL ELECTROPHORESIS (DAY 2)
Sharpie marking pen
gloves
250 ml flask
balance, spatula and weigh boats
agarose
microwave oven
heating block set at 50 C (1.5 mL tubes)
hot gloves
1x TAE electrophoresis buffer (dilute
from 50x, if needed)
100 ml graduated cylinder
dH2O
horizontal gel electrophoresis rig
automatic micropipetters and tips
used tip container
Kim wipes
1.5 ml microcentrifuge tubes
microfuge tube rack
D.C. power supply