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Lab meeting 2013.05.13

Lab meeting 2013.05.13. Nguyen Thi Dai Trang. U2AF1. 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

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Lab meeting 2013.05.13

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  1. Lab meeting 2013.05.13 Nguyen Thi Dai Trang

  2. U2AF1 Electroporation of K562, Hela, IM9 • Protocol 1. 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

  3. 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 Source: 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, 1x107

  5. SRSF2 Step 3: cDNA SRSF2 digested with NheI, XhoI cDNA of SRSF2 gene was composed of 231 aa and 714 bp.  Source: http://tools.neb.com/NEBcutter2/showdig.php?name=e051c50b-SRSF2-cloning_seq_C-

  6. 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 (40-46ng/ µl) 5 µl • Distilled water 37 µl • Total: 50 µl • Incubation time: overnight • Incubation temp: 37 °C After digested by RE NheI & XhoI K562 Hela IM9 Digested cDNA U2AF1 (546 bp) 708 bp Ready for ligation into pcDNA3.1

  7. SRSF2 Step 3: pcDNA3.1 digested with NheI, XhoI Source: http://tools.neb.com/NEBcutter2/showdig.php?name=a4b78c98-pcDNA_3.1_seq

  8. 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 • pcDNA3.1 (55ng/ µl) 1 µl • Distilled water 41 µl • Total: 50 µl • Incubation time: overnight • Incubation temp: 37 °C Digested pcDNA 3.1 (5338bp) 1kb plus DNA ladder 5000 bp Ready for ligation with cDNA SRSF2

  9. SRSF2 Step 4: Ligate into expression vector pcDNA3.1 • Ligation Reaction • 2X Rapid Ligation buffer, T4 DNA ligase 5 µl • pcDNA3.1 vector (55ng/ 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

  10. This week’s plan SRSF2 • Ligate the insert into the pcDNA3.1 vector and transform into E. coli. • Selecttransformants 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 U2AF1

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