AlanFung:LabNotes/DNA/2009-8-18: Difference between revisions
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==Digestion with MmeI== | ==Digestion with MmeI== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"| | | align="center" style="background:#f0f0f0;"| | ||
| align="center" style="background:#f0f0f0;"|'''PGP4''' | | align="center" style="background:#f0f0f0;"|'''PGP4''' | ||
| align="center" style="background:#f0f0f0;"|'''PGP6''' | | align="center" style="background:#f0f0f0;"|'''PGP6''' | ||
| align="center" style="background:#f0f0f0;"|'''PGP10''' | | align="center" style="background:#f0f0f0;"|'''PGP10''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''5012A''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''5012B''' | ||
|- | |- | ||
| Sample||20||20||20||10||10 | | Sample||20||20||20||10||10 | ||
Line 12: | Line 12: | ||
| 10X NEBuffer 4||4||4||4||3||3 | | 10X NEBuffer 4||4||4||4||3||3 | ||
|- | |- | ||
| 1mM SAM (fresh)|| | | 1mM SAM (fresh)||8||8||8||6||6 | ||
|- | |- | ||
| 2U/ul MmeI||1||1||1||1||1 | | 2U/ul MmeI||1||1||1||1||1 | ||
|- | |- | ||
| ddH20|| | | ddH20||7||7||7||10||10 | ||
|- | |- | ||
| Total||40||40||40||30||30 | | Total||40||40||40||30||30 | ||
Line 24: | Line 24: | ||
1mM SAM: 32mM SAM 1ul + 31ul ddH2O. | 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. | ||
37C 2h. | 37C 2h. | ||
*Qiaquick column purification. Elute in 30ul EB. | |||
===quantification with qPCR=== | |||
*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 | |||
{| {{table}} | |||
|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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''nM''' | |||
| align="center" style="background:#f0f0f0;"|'''sample ul''' | |||
| align="center" style="background:#f0f0f0;"|'''Tris-CL ul''' | |||
| align="center" style="background:#f0f0f0;"|'''HP3''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|- | |||
| | |||
|} |
Latest revision as of 16:33, 20 August 2009
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 |