02/02/2015
PCR of gRNA from Luc14 and GAL4-EED+dox samples, g034
Cell type
|
treatment
|
rep
|
Concentration
|
ul for 200ng
|
ul for 800ng
|
Luc14 |
g034 |
1 |
71.774 |
2.79 |
11.15
|
Luc14 |
g034 |
2 |
117.513 |
1.70 |
6.81
|
Luc14 |
g034 |
3 |
108.009 |
1.85 |
7.41
|
GAL4-EED +dox |
g034 |
1 |
86.013 |
2.33 |
9.30
|
GAL4-EED +dox |
g034 |
2 |
105.127 |
1.90 |
7.61
|
GAL4-EED +dox |
g034 |
3 |
122.48 |
1.63 |
6.53
|
Mastermix 1
'
|
1rxn
|
25.5
|
H20 |
30.8 |
785.4
|
HF 5X MasterMix |
10 |
255
|
dNTPs |
0.2 |
5.1
|
FP |
2.5 |
63.75
|
RP |
2.5 |
63.75
|
Total |
46 |
1173
|
Mastermix 2
'
|
1rxn
|
4.2rxns
|
MM1 |
46 |
193.2
|
Template |
2 |
8.4
|
DMSO |
1.5 |
6.3
|
Phusion |
0.5 |
2.1
|
Total |
50 |
205
|
Transfer 50μL of MM2 into each of 4-PCR tubes.
PCR setup
PCR
- 98°C for 3 min
- 30 cycles
- 98°C for 10s
- 69.5°C for 30s
- 72°C for 60s
- 72°C for 10 min
- 4°C forever
Purified with Sigma genelute PCR clean up kit. Yields aren't as high as I'd like but I can work with it, especially if it reduces my background.
Cell type
|
treatment
|
rep
|
260
|
280
|
260/280
|
ng/µL
|
Luc14 |
g034 |
1 |
0.029 |
0.017 |
1.75 |
29.049
|
Luc14 |
g034 |
2 |
0.029 |
0.016 |
1.839 |
28.848
|
Luc14 |
g034 |
3 |
0.039 |
0.021 |
1.835 |
39.051
|
GAL4-EED +dox |
g034 |
1 |
0.039 |
0.021 |
1.841 |
38.915
|
GAL4-EED +dox |
g034 |
2 |
0.038 |
0.019 |
2.028 |
37.778
|
GAL4-EED +dox |
g034 |
3 |
0.042 |
0.022 |
1.914 |
42.041
|
|