Lab meeting 2013.04.08

• Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl)

Linearize pcDNA3.1+cDNA U2AF1 by ScaI

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 digestedNeed 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 0.2510x buffer LA tag 5dNTP 4cDNA 2Primer F 1

R 1DW 36.75Total 50

98  °C : 98  °C : 62  °C : 72  °C : 20  °C5min : 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

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

dNTP 4

cDNA (250 ng/µl) 2

Primer F (20pmol/ul) 1

R(pmol/ul) 1

D.W 28

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 tubeHeat to 94°C 3’Add LA tag fraction during first annealing/extension step.

94  °C : 94  °C : 59  °C : 68  °C : 72°C :20  °C3’ : 30” :30” : 1’ : 5’ : --

35 cycles

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

94  °C : 94  °C : 60  °C : 68  °C : 72°C :20  °C3’ : 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

cDNA SRSF2 Amplify 662 bp by Ex tag Takara(Gradient, using 5XCES)

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

94  °C : 94  °C : gradient  °C : 68  °C : 72°C :20  °C3’ : 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