AlanFung:LabNotes/Sequencing/2009-11-19

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Quantification of New amplified Phi-X and all libraries[edit]

  • Dilute all samples by 10 fold
Samples
PhiX-Stock CVF PGP4 PGP10 PGP8C
PhiX-Stock CVF PGP4 PGP10 PGP8C
PhiX-Stock 1-7 PGP1 PGP6 NA12878 PGP8D
PhiX-Stock 1-7 PGP1 PGP6 NA12878 PGP8D
PhiX 8 PGP1 PGP8 PGP8A PGP8E
PhiX 8 PGP1 PGP8 PGP8A PGP8E
CVI 9 PGP1 PGP9 PGP8B N12
CVI 9 PGP1 PGP9 PGP8B N12
  • split every 50ul rxn into 2 wells
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min
X20 X1.1
PhiX Control DNA (10nM) 1.000
Syb_FP5 (100uM) 0.200 4.000 4.400
Syb_RP7 (100uM) 0.200 4.000 4.400
2X Phusion 25.000 500.000 550.000
50X SYBR Green I 0.200 4.000 4.400
H20 24.000 480.000 528.000

Results[edit]

Result
PhiX 9.112 Cycle Difference 2^ x10 (nM)
Amp PhiX 6.051 3.062 8.348 83.484
CVI 6.877 2.236 4.709 47.093
CVF 8.155 0.958 1.942 19.419
1-7 PGP1 8.346 0.766 1.701 17.011
8 PGP1 6.510 2.603 6.073 60.734
9 PGP1 7.244 1.868 3.650 36.503
N12 7.915 1.198 2.293 22.934
PGP8B 11.776 2.664 6.339 1.577

Dilution[edit]

Dilute to 10nM Sample (ul) ddH2O
Amp PhiX 5.000 36.742
CVI 5.000 18.546
CVF 5.000 4.710
1-7 PGP1 5.000 3.506
8 PGP1 5.000 25.367
9 PGP1 5.000 13.251
N12 5.000 6.467
PGP8B 5.000 2.885 1nM