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Lab meeting 2013.04.08
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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
![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](http://images.slideplayer.com/25/7925325/slides/slide_2.jpg "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")
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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
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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
![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](http://images.slideplayer.com/25/7925325/slides/slide_4.jpg "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")
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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
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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
![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.](http://images.slideplayer.com/25/7925325/slides/slide_6.jpg "94 °C : 94 °C : 59 °C : 68 °C : 72 °C :20 °C 3’: 30 :30 : 1’: 5’: cycles.")
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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
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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
![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](http://images.slideplayer.com/25/7925325/slides/slide_8.jpg "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")
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