1. Lab meeting 2013.05.13 Nguyen Thi Dai Trang

2. Electroporation of K562, Hela, IM9  Protocol 1. 2x10 6 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

3. RESULTS All K562, Hela, IM9 cells die Assumed reasons: + capacitance, voltage, cell density (3µF, 800 V, 2x10 6 ) + kit for purification DNA enclosed toxins for mamalian cells U2AF1 Source: http://www.biorad-ads.com/transfection_protocols/index.html?source_wt=transfectionprotocols_surl http://www.biorad-ads.com/transfection_protocols/index.html?source_wt=transfectionprotocols_surl

4. Solution  Try 1 more time using following condition: For Hela: 166V, 500µF, 1x10 7

5. 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. XhoIC’TCGA,G NheIG’CTAG,C EnzymeSpecificity SRSF2

6. 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

7. Step 3: pcDNA3.1 digested with NheI, XhoI Source: http://tools.neb.com/NEBcutter2/showdig.php?name=a4b78c98-pcDNA_3.1_seq XhoIC’TCGA,G NheIG’CTAG,C EnzymeSpecificity SRSF2

8. 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

9. 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

10. 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