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Lab meeting 2013.04.08

Lab meeting 2013.04.08. Linearize pcDNA3.1+cDNA U2AF1 by ScaI. Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl). ScaI 1µl 10X NEB buffer (No.3) 5µl BSA 2µl pcDNA3.1+cDNA (1µg/µl) 1µl D.W 41µl Total 50µl. Incubation time: O/N Incubation temp: 37 °C. Not completely digested

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Lab meeting 2013.04.08

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  1. Lab meeting 2013.04.08

  2. Linearize pcDNA3.1+cDNA U2AF1 by ScaI • Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl) ScaI 1µl 10X NEB buffer (No.3) 5µl BSA 2µl pcDNA3.1+cDNA (1µg/µl) 1µl D.W 41µl 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) Product length: 662 bp LA Tag 0.25 10x buffer LA tag 5 dNTP 4 cDNA 2 Primer F 1 R 1 DW 36.75 Total 50 30 cycles 98  °C : 98  °C : 62  °C : 72  °C : 20  °C 5min : 10 sec :30 sec : 5 min : -- • Prepare mastermix w/o Ex tag • add template • Put in thermocycler 98  °C 5 min • add Ex-tag

  6. cDNA SRSF2 Amplify 662 bp by Ex tag Takara (hot-start method) 1/5 LA tag fraction Ex tag 0.3 Buffer Ex tag 1 D.W 8.7 Total 10µl 4/5 [template+ primer] fraction Buffer Ex tag 4 dNTP 4 cDNA (250 ng/µl) 2 Primer F (20pmol/ul) 1 R(pmol/ul) 1 D.W 28 Total 40µl 35 cycles 94  °C : 94  °C : 59  °C : 68  °C : 72°C :20  °C 3’ : 30” :30” : 1’ : 5’ : -- • Put [template + primer] fraction in the tube • Heat to 94°C 3’ • Add LA tag fraction during first annealing/extension step. LadderK562 Hela IM9

  7. cDNA SRSF2 Amplify 662 bp by Ex tag Takara PCR reation Buffer Ex tag 5 dNTP 4 cDNA (250 ng/µl) 2 Primer F (20pmol/ul) 1 R(pmol/ul) 1 Ex-tag 0,3 D.W 36,7 Total 50µl 35 cycles 1500bp 1000bp 500bp K562 Hela IM9 Ladder 94  °C : 94  °C : 60  °C : 68  °C : 72°C :20  °C 3’ : 30” :30” : 1’ : 5’ : -- • No target product was observed • Do gradient annealing temp to find optimal degree for annealing temp

  8. cDNA SRSF2 Amplify 662 bp by Ex tag Takara (Gradient, using 5XCES) 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 PCR reation Buffer Ex tag 5 dNTP 4 cDNA (250 ng/µl) 2 Primer F (20pmol/ul) 1 R(pmol/ul) 1 Ex-tag 0,3 5XCES 5 D.W 31.7 Total 50µl 35 cycles 94  °C : 94  °C : gradient  °C : 68  °C : 72°C :20  °C 3’ : 30” :30” : 1’ : 5’ : -- Grad K562 54 °C 56 °C 58 °C 60 °C 62 °C Ladder • 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 1000bp 500bp

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