AlanFung:LabNotes/Sequencing/2009-12-5: Difference between revisions

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>Alan6017518
>Alan6017518
 
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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