09/20/2014
Ran gel of gradient +/- DMSO PCR reactions from yesterday
Seems obvious that DMSO is necessary for PCR to work. Interesting that the higher annealing temperatures work better than lower ones. Start seeing two bands closer to 56°C.
PCR of gRNA 20 samples
Samples
|
gRNA 20 + dox 1
|
gRNA 20 + dox 2
|
gRNA 20 - dox 1
|
gRNA 20 - dox 2
|
gRNA 20 Luc 14
|
Master mix 1
'
|
1 rxn
|
5.5 rxns
|
water |
23.5 |
129.25
|
5x buffer |
10 |
55
|
dNTPs |
1 |
5.5
|
FP |
2.5 |
13.75
|
RP |
2.5 |
13.75
|
DMSO |
1.5 |
8.25
|
|
41 |
|
add 41ul of MM 1 to each tube
add 4ul of each template to each tube (400ng)
'
|
1 rxn
|
5.5 rxns
|
H20 |
4.5 |
24.75
|
Phusion |
0.5 |
2.75
|
add 5 ul of MM 2 to each tube
PCR
- 98°C for 3 min
- 36 cycles
- 98°C for 10s
- 66°C for 30s
- 72°C for 60s
- 72°C for 10 min
- 4°C forever
|