AlanFung:LabNotes/Sequencing/2009-12-5: Difference between revisions
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>Alan6017518 |
>Alan6017518 |
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(16 intermediate revisions by the same user not shown) | |||
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|- | |- | ||
| Total||||||982.08 | | Total||||||982.08 | ||
|- | |||
| | |||
|} | |||
*Dilute sample, 1ul in 9ul ddh2o | |||
*mix 2ul 1/10 sample with 98ul mix | |||
*aliquot 24ul to each well | |||
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min | |||
{| {{table}} | |||
| ||||||||||Average||Difference||2^||nM | |||
|- | |||
| Amp Phi-X (1nM)||9.833||9.899||9.819||9.990||9.89||0|||| | |||
|- | |||
| PGP4||9.471||9.522||9.562||9.706||9.57||0.32||1.25||1.25 | |||
|- | |||
| PGP6||10.667||10.911||11.011||||10.86||0.97||1.96||0.51 | |||
|- | |||
| PGP8||9.719||9.813||10.088||10.166||9.95||0.06||1.04||0.96 | |||
|- | |||
| PGP9||9.663||9.710||9.751||||9.71||0.18||1.13||1.13 | |||
|- | |||
| PGP8D||5.250||5.262||5.287||||5.27||4.62||24.65||24.65 | |||
|- | |||
| PGP8E||5.067||5.143||5.168||||5.13||4.76||27.17||27.17 | |||
|- | |||
| Dpn-CV-F2 (1/10)||8.303||8.403||8.663||8.666||8.51||1.38||2.6||26 | |||
|- | |||
| Dpn-CV-iF2 (1/10)||9.824||9.962||10.000||||9.93||0.04||1.03||9.74 | |||
|- | |||
| | |||
|} | |||
==Amplify PGP Libraries (5 Cycles)== | |||
{| {{table}} | |||
| ||F||R | |||
|- | |||
| 10uM Solexa_PCR_up||30.8||30.8 | |||
|- | |||
| 10uM AmpR6.3Sol||0||30.8 | |||
|- | |||
| 10uM AmpF6.3Sol||30.8||0 | |||
|- | |||
| 2X Phusion||770||770 | |||
|- | |||
| 50X SYBR Green I||6.16||6.16 | |||
|- | |||
| H2O||554.4||554.4 | |||
|- | |||
| Total||1392.16||1392.16 | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| Setup|| | |||
|- | |||
| PGP4 F||PGP10 F | |||
|- | |||
| PGP4 R||PGP10 R | |||
|- | |||
| PGP6 F||PGP8C F | |||
|- | |||
| PGP6 R||PGP8C R | |||
|- | |||
| PGP8 F||NA12878 F | |||
|- | |||
| PGP8 R||NA12878 R | |||
|- | |||
| PGP9 F|| | |||
|- | |||
| PGP9 R|| | |||
|- | |||
| | |||
|} | |||
PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. | |||
*Minelute with 12ul | |||
==qPCR Quantification== | |||
{| {{table}} | |||
| Setup|||||| | |||
|- | |||
| Amp Phi-X (1nM)||PGP6||PGP9||PGP8C | |||
|- | |||
| Amp Phi-X (1nM)||PGP6||PGP9||PGP8C | |||
|- | |||
| Amp Phi-X (1nM)||PGP6||PGP9||PGP8C | |||
|- | |||
| Amp Phi-X (1nM)||PGP6||PGP9||PGP8C | |||
|- | |||
| PGP4||PGP8||PGP10||NA12878 | |||
|- | |||
| PGP4||PGP8||PGP10||NA12878 | |||
|- | |||
| PGP4||PGP8||PGP10||NA12878 | |||
|- | |||
| PGP4||PGP8||PGP10||NA12878 | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| ||||X16||X1.1 | |||
|- | |||
| DNA||2|||| | |||
|- | |||
| Syb_FP5 (100uM)||0.2||3.2||3.52 | |||
|- | |||
| Syb_RP7 (100uM)||0.2||3.2||3.52 | |||
|- | |||
| 2X Phusion||25||400||440 | |||
|- | |||
| 50X SYBR Green I||0.2||3.2||3.52 | |||
|- | |||
| H20||24||384||422.4 | |||
|- | |||
| Total||||||872.96 | |||
|- | |||
| | |||
|} | |||
*Dilute sample, 1ul in 9ul ddh2o | |||
*mix 2ul 1/10 sample with 98ul mix | |||
*aliquot 24ul to each well | |||
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min | |||
{| {{table}} | |||
| Dilute to 10nM||nM||To 10nM||ddH20||Sample|| | |||
|- | |||
| Dpn-CV-F2 ||26.000||10.000||8.000||5.000|| | |||
|- | |||
| PGP8D||24.650||10.000||7.325||5.000|| | |||
|- | |||
| PGP8E||27.170||10.000||8.585||5.000|| | |||
|- | |||
| PGP8||27.484||10.000||8.742||5.000|| | |||
|- | |||
| |||||||||| | |||
|- | |||
| Dilute to 1nM||nM||sample ul||Tris-CL ul||HP3||total | |||
|- | |||
| Dpn-CV-iF2 ||9.740||2.000||16.506||0.974||19.480 | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| ||PGP4||PGP6||PGP10||PGP9||PGP8C||NA12878 | |||
|- | |||
| ||5.936||6.343||5.427||6.815||6.229||5.857 | |||
|- | |||
| ||5.939||6.471||5.602||6.897||6.342||5.907 | |||
|- | |||
| ||6.024||7.001||5.725|||||| | |||
|- | |||
| ||||7.048||5.972|||||| | |||
|- | |||
| ||5.966||6.716||5.682||6.856||6.286||5.882 | |||
|- | |||
| Difference||4.884||4.134||5.169||3.994||4.565||4.968 | |||
|- | |||
| 2^||29.521||17.560||35.964||15.934||23.662||31.298 | |||
|- | |||
| |||||||||||| | |||
|- | |||
| Dilute to 10nM||nM||To 10nM||ddH20||Sample||total|| | |||
|- | |||
| PGP4||29.521||10.000||9.761||5.000||14.76|| | |||
|- | |||
| PGP6||17.560||10.000||3.780||5.000||8.78|| | |||
|- | |||
| PGP9||15.934||10.000||2.967||5.000||7.97|| | |||
|- | |||
| PGP10||35.964||10.000||12.982||5.000||17.98|| | |||
|- | |||
| PGP8C||23.662||10.000||6.831||5.000||11.83|| | |||
|- | |||
| NA12878||31.298||10.000||10.649||5.000||15.65|| | |||
|- | |- | ||
| | | | ||
|} | |} |
Latest revision as of 19:28, 7 December 2009
qPCR Quantification[edit]
PGP4 PGP6 PGP8 PGP9 PGP8D PGP8E Dpn-CV-iF2 Dpn-CV-F2[edit]
Setup | ||||
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8E | Dpn-CV-iF2 (1/10) |
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8E | Dpn-CV-iF2 (1/10) |
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8E | Dpn-CV-iF2 (1/10) |
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8E | Dpn-CV-iF2 (1/10) |
PGP4 | PGP8 | PGP8D | Dpn-CV-F2 (1/10) | |
PGP4 | PGP8 | PGP8D | Dpn-CV-F2 (1/10) | |
PGP4 | PGP8 | PGP8D | Dpn-CV-F2 (1/10) | |
PGP4 | PGP8 | PGP8D | Dpn-CV-F2 (1/10) | |
x18 | *1.1 | ||
DNA | 2 | ||
Syb_FP5 (100uM) | 0.2 | 3.6 | 3.96 |
Syb_RP7 (100uM) | 0.2 | 3.6 | 3.96 |
2X Phusion | 25 | 450 | 495 |
50X SYBR Green I | 0.2 | 3.6 | 3.96 |
H20 | 24 | 432 | 475.2 |
Total | 982.08 | ||
- Dilute sample, 1ul in 9ul ddh2o
- mix 2ul 1/10 sample with 98ul mix
- aliquot 24ul to each well
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min
Average | Difference | 2^ | nM | |||||
Amp Phi-X (1nM) | 9.833 | 9.899 | 9.819 | 9.990 | 9.89 | 0 | ||
PGP4 | 9.471 | 9.522 | 9.562 | 9.706 | 9.57 | 0.32 | 1.25 | 1.25 |
PGP6 | 10.667 | 10.911 | 11.011 | 10.86 | 0.97 | 1.96 | 0.51 | |
PGP8 | 9.719 | 9.813 | 10.088 | 10.166 | 9.95 | 0.06 | 1.04 | 0.96 |
PGP9 | 9.663 | 9.710 | 9.751 | 9.71 | 0.18 | 1.13 | 1.13 | |
PGP8D | 5.250 | 5.262 | 5.287 | 5.27 | 4.62 | 24.65 | 24.65 | |
PGP8E | 5.067 | 5.143 | 5.168 | 5.13 | 4.76 | 27.17 | 27.17 | |
Dpn-CV-F2 (1/10) | 8.303 | 8.403 | 8.663 | 8.666 | 8.51 | 1.38 | 2.6 | 26 |
Dpn-CV-iF2 (1/10) | 9.824 | 9.962 | 10.000 | 9.93 | 0.04 | 1.03 | 9.74 | |
Amplify PGP Libraries (5 Cycles)[edit]
F | R | |
10uM Solexa_PCR_up | 30.8 | 30.8 |
10uM AmpR6.3Sol | 0 | 30.8 |
10uM AmpF6.3Sol | 30.8 | 0 |
2X Phusion | 770 | 770 |
50X SYBR Green I | 6.16 | 6.16 |
H2O | 554.4 | 554.4 |
Total | 1392.16 | 1392.16 |
Setup | |
PGP4 F | PGP10 F |
PGP4 R | PGP10 R |
PGP6 F | PGP8C F |
PGP6 R | PGP8C R |
PGP8 F | NA12878 F |
PGP8 R | NA12878 R |
PGP9 F | |
PGP9 R | |
PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.
- Minelute with 12ul
qPCR Quantification[edit]
Setup | |||
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8C |
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8C |
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8C |
Amp Phi-X (1nM) | PGP6 | PGP9 | PGP8C |
PGP4 | PGP8 | PGP10 | NA12878 |
PGP4 | PGP8 | PGP10 | NA12878 |
PGP4 | PGP8 | PGP10 | NA12878 |
PGP4 | PGP8 | PGP10 | NA12878 |
X16 | X1.1 | ||
DNA | 2 | ||
Syb_FP5 (100uM) | 0.2 | 3.2 | 3.52 |
Syb_RP7 (100uM) | 0.2 | 3.2 | 3.52 |
2X Phusion | 25 | 400 | 440 |
50X SYBR Green I | 0.2 | 3.2 | 3.52 |
H20 | 24 | 384 | 422.4 |
Total | 872.96 | ||
- Dilute sample, 1ul in 9ul ddh2o
- mix 2ul 1/10 sample with 98ul mix
- aliquot 24ul to each well
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min
Dilute to 10nM | nM | To 10nM | ddH20 | Sample | |
Dpn-CV-F2 | 26.000 | 10.000 | 8.000 | 5.000 | |
PGP8D | 24.650 | 10.000 | 7.325 | 5.000 | |
PGP8E | 27.170 | 10.000 | 8.585 | 5.000 | |
PGP8 | 27.484 | 10.000 | 8.742 | 5.000 | |
Dilute to 1nM | nM | sample ul | Tris-CL ul | HP3 | total |
Dpn-CV-iF2 | 9.740 | 2.000 | 16.506 | 0.974 | 19.480 |
PGP4 | PGP6 | PGP10 | PGP9 | PGP8C | NA12878 | |
5.936 | 6.343 | 5.427 | 6.815 | 6.229 | 5.857 | |
5.939 | 6.471 | 5.602 | 6.897 | 6.342 | 5.907 | |
6.024 | 7.001 | 5.725 | ||||
7.048 | 5.972 | |||||
5.966 | 6.716 | 5.682 | 6.856 | 6.286 | 5.882 | |
Difference | 4.884 | 4.134 | 5.169 | 3.994 | 4.565 | 4.968 |
2^ | 29.521 | 17.560 | 35.964 | 15.934 | 23.662 | 31.298 |
Dilute to 10nM | nM | To 10nM | ddH20 | Sample | total | |
PGP4 | 29.521 | 10.000 | 9.761 | 5.000 | 14.76 | |
PGP6 | 17.560 | 10.000 | 3.780 | 5.000 | 8.78 | |
PGP9 | 15.934 | 10.000 | 2.967 | 5.000 | 7.97 | |
PGP10 | 35.964 | 10.000 | 12.982 | 5.000 | 17.98 | |
PGP8C | 23.662 | 10.000 | 6.831 | 5.000 | 11.83 | |
NA12878 | 31.298 | 10.000 | 10.649 | 5.000 | 15.65 | |