Lab meeting

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

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

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

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

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’: cycles

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’: cycles K562 Hela IM9 Ladder  No target product was observed  Do gradient annealing temp to find optimal degree for annealing temp 1500bp 1000bp 500bp

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’: 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 K bp 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