1. Lab meeting 2013.04.08

2. Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl) Linearize pcDNA3.1+cDNA U2AF1 by ScaI ScaI1µl 10X NEB buffer (No.3)5µl BSA2µl pcDNA3.1+cDNA (1µg/µl)1µl D.W41µl Total 50µl Incubation time: O/N Incubation temp: 37 °C  Not completely digested  Need to use new enzyme

3. Neomycin Antibiotic sensitivities of Hela, THP-1, K562 All cells grow happily at 0, 300, 400, 500, 1000, 1200 μg/ml  need to use new kind of antibiotic to select cells  puromycin plasmid co-transfection

4. pCAGIPuro plasmid  Do Puromycin antibiotic sensitivities of Hela, K562, IM9  Do plasmid preparation of PCAGIPuro-midi kit  Linearize by PvuI  Co-transfection of [pCDNA3.1+cDNA U2AF1 ] and pCAGIPuro to cell lines Hela, K562, IM9  Expected time: 2 weeks

5. cDNA SRSF2 Amplify 662 bp by Ex tag Takara (hot-start method) LA Tag 0.25 10x buffer LA tag5 dNTP4 cDNA2 Primer F1 R1 DW36.75 Total 50 98 °C : 98 °C : 62 °C : 72 °C : 20 °C 5min: 10 sec :30 sec: 5 min: -- 30 cycles -Prepare mastermix w/o Ex tag - add template -Put in thermocycler 98 °C 5 min - add Ex-tag Product length: 662 bp

6. cDNA SRSF2 Amplify 662 bp by Ex tag Takara (hot-start method) Ladder K562 Hela IM9 4/5 [template+ primer] fraction Buffer Ex tag 4 dNTP4 cDNA (250 ng/µl)2 Primer F (20pmol/ul)1 R(pmol/ul)1 D.W28 Total 40µl 1/5 LA tag fraction Ex tag 0.3 Buffer Ex tag 1 D.W8.7 Total 10µl  Put [template + primer] fraction in the tube  Heat to 94°C 3’  Add LA tag fraction during first annealing/extension step. 94 °C : 94 °C : 59 °C : 68 °C : 72 °C :20 °C 3’: 30” :30”: 1’: 5’: -- 35 cycles

7. cDNA SRSF2 Amplify 662 bp by Ex tag Takara PCR reation Buffer Ex tag 5 dNTP4 cDNA (250 ng/µl)2 Primer F (20pmol/ul)1 R(pmol/ul)1 Ex-tag0,3 D.W36,7 Total 50µl 94 °C : 94 °C : 60 °C : 68 °C : 72 °C :20 °C 3’: 30” :30”: 1’: 5’: -- 35 cycles K562 Hela IM9 Ladder  No target product was observed  Do gradient annealing temp to find optimal degree for annealing temp 1500bp 1000bp 500bp

8. cDNA SRSF2 Amplify 662 bp by Ex tag Takara (Gradient, using 5XCES) PCR reation Buffer Ex tag 5 dNTP4 cDNA (250 ng/µl)2 Primer F (20pmol/ul)1 R(pmol/ul)1 Ex-tag0,3 5XCES5 D.W31.7 Total 50µl 94 °C : 94 °C : gradient °C : 68 °C : 72 °C :20 °C 3’: 30” :30”: 1’: 5’: -- 35 cycles 5XCES = [2.7M betaine, 6.7 mM DTT+ 6,7% DMSO + 55µg/mL BSA]  use for amplify products which have high GC content and reducing secondary structure 54 °C 56 °C 58 °C 60 °C 62 °C Ladder Grad K562 1000bp 500bp  Result at 60 °C in this case is d/f with one in previous slide at 60 °C  5XCES maybe use from now on to prevent unspecific bands  Little small band of target product was observed  Many unspecific band was removed at 62 °C  Try again at 62 °C and 64 °C