Post on 02-Jan-2016
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