Lab meeting 2013.05.13

Nguyen Thi Dai Trang

Electroporation of K562, Hela, IM9

Protocol1. 2x106 cells

2. PBS wash 2 times

3. Suspend in 90µl PBS

4. Add 10µl linear vector (10µg). Total volume now is 100µl

5. Incubate on ice 10 min

6. Electroporation 3µF, 800 V

7. Spread on 100 mm disk with full RPMI

8. 48 hours, add Puromycin to selection

U2AF1

RESULTS

• All K562, Hela, IM9 cells die

• Assumed reasons:

• + capacitance, voltage, cell density (3µF, 800 V, 2x106)

+ kit for purification DNA enclosed toxins for mamalian cells

U2AF1

Source: http://www.biorad-ads.com/transfection_protocols/index.html?source_wt=transfectionprotocols_surl

Solution

Try 1 more time using following condition:

• For Hela: 166V, 500µF, 1x107

Step 3: cDNA SRSF2 digested with NheI, XhoI

Source: http://tools.neb.com/NEBcutter2/showdig.php?name=e051c50b-SRSF2-cloning_seq_C-

cDNA of SRSF2 gene was composed of 231 aa and 714 bp. 

XhoI C’TCGA,G

NheI G’CTAG,C

Enzyme Specificity

SRSF2

Step 3: Restriction Endonuclease NheI, XhoI

Restriction Digestion for Ligation Reaction

NheI 1 µl

XhoI 1 µl

10X NEBuffer (1X) 5 µl

BSA 1 µl

cDNA SRSF2 (40-46 ng/ µl) 5 µl

Distilled water 37 µl

Total: 50 µl

• Incubation time: overnight

• Incubation temp: 37 °C

After digested by RE NheI & XhoI

Digested cDNA U2AF1

(546 bp)

K562 Hela IM9

708 bp

Ready for ligation into pcDNA3.1

SRSF2

Step 3: pcDNA3.1 digested with NheI, XhoI

Source: http://tools.neb.com/NEBcutter2/showdig.php?name=a4b78c98-pcDNA_3.1_seq

XhoI C’TCGA,G

NheI G’CTAG,C

Enzyme Specificity

SRSF2

Step 3: Restriction Endonuclease NheI, XhoI

Ready for ligation with cDNA SRSF2

1kb plus DNA ladder

5000 bp

Digested pcDNA 3.1(5338bp)

Restriction Digestion for Ligation Reaction

NheI 1 µl

XhoI 1 µl

10X NEBuffer (1X) 5 µl

BSA 1 µl

pcDNA3.1 (55 ng/ µl) 1 µl

Distilled water 41 µl

Total: 50 µl

• Incubation time: overnight

• Incubation temp: 37 °C

SRSF2

Step 4: Ligate into expression vector pcDNA3.1

Ligation Reaction

2X Rapid Ligation buffer, T4 DNA ligase 5 µl

pcDNA3.1 vector (55 ng/ µl) 1 µl

cDNA U2AF1 (55 ng/ µl) 3 µl

T4 DNA ligase (3 units/µl) 1 µl

Distilled water 10 µl

Total: 20 µl

• Incubation time: overnight

• Incubation temp: 4°C

SRSF2

This week’s plan

• Ligate the insert into the pcDNA3.1 vector and transform into E. coli.

• Select transformants on LB plates containing 100 μg/ml ampicillin.

• Analyze and select transformants for the presence of insert by PCR, restriction digestion and sequencing.

• Try electroporation again with Hela

SRSF2

U2AF1