Revision as of 16:33, 20 August 2009 by >Alan6017518
Digestion with MmeI[edit]
|
PGP4
|
PGP6
|
PGP10
|
5012A
|
5012B
|
Sample |
20 |
20 |
20 |
10 |
10
|
10X NEBuffer 4 |
4 |
4 |
4 |
3 |
3
|
1mM SAM (fresh) |
8 |
8 |
8 |
6 |
6
|
2U/ul MmeI |
1 |
1 |
1 |
1 |
1
|
ddH20 |
7 |
7 |
7 |
10 |
10
|
Total |
40 |
40 |
40 |
30 |
30
|
|
1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
37C 2h.
- Qiaquick column purification. Elute in 30ul EB.
quantification with qPCR[edit]
- Dilute the PhiX lib(10nM) to 1nM, 0.1nM, 0.025nM, 0.005nM
- Dilute the samples to 1:10 and 1:50 ratio
- 1:10-Add 1ul of sample to 9ul H20
- 1:50-Add 1ul of 1:10 sample to 4ul H20
Syb_FP5: ATGATACGGCGACCACCGAG
Syb_RP7: CAAGCAGAAGACGGCATACGAG
1nM PhiX |
1:10 PGP4 |
1:10 PGP10 |
1:10 50192B
|
1nM PhiX |
1:10 PGP4 |
1:10 PGP10 |
1:10 50192B
|
0.1nM PhiX |
1:50 PGP4 |
1:50 PGP10 |
1:50 50192B
|
0.1nM PhiX |
1:50 PGP4 |
1:50 PGP10 |
1:50 50192B
|
0.025nM PhiX |
1:10 PGP6 |
1:10 50192A |
|
0.025nM PhiX |
1:10 PGP6 |
1:10 50192A |
|
0.005nM PhiX |
1:50 PGP6 |
1:50 50192A |
|
0.005nM PhiX |
1:50 PGP6 |
1:50 50192A |
|
|
|
X29 |
|
Template |
1ul |
|
|
2X iProof Mix |
25 |
725 |
|
SYB_RP7 (100uM) |
0.2 |
5.8 |
|
SYB_FP5 (100uM) |
0.2 |
5.8 |
|
H2O |
24 |
696 |
|
50X SYBR I |
0.2 |
5.8 |
|
Toal |
|
1438.4 |
|
|
98C 30sec -> 30 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold.
- stopped when curve reached plateau
'
|
nM
|
sample ul
|
Tris-CL ul
|
HP3
|
total
|
PGP4 |
1.71 |
10 |
6.25 |
0.86 |
17.1
|
PGP6 |
1.21 |
10 |
1.5 |
0.61 |
12.1
|
PGP10 |
1.56 |
10 |
4.82 |
0.78 |
15.6
|
50192A |
2.44 |
10 |
13.18 |
1.22 |
24.4
|
50192B |
1.4 |
10 |
3.3 |
0.7 |
14
|
|
|
|
|
|
|
PGP6 |
1.21 |
20 |
2.99 |
1.21 |
24.2
|
|