AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4

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Revision as of 18:41, 4 December 2009 by >Alan6017518 (→‎Prepare Master Mix)
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qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)

  • Start with 10ng template
Sample Sample Concentration ng/ul To get 10ng 1/10 Dilution ng/ul To get 10ng
NA12878 17.6 0.57
NA18507 12.4 0.81
NA20431 50.6 5.06 1.98
CV-Fibr 4.84 2.07
CV-iPS-B 7.27 1.38
CV-iPS-F 6.91 1.45
Jurkat 55.4 5.54 1.81
Jurkat (old) 104 10.4 0.96
  • Mix 50/50 old/new Jurkat to make up 10ng
New: 0.905ul
Old: 0.48ul
Sample Volume Make it up to 8ul
NA12878 0.57 7.43
NA18507 0.81 7.19
NA20431 1.98 6.02
CV-Fibr 2.07 5.93
CV-iPS-B 1.38 6.62
CV-iPS-F 1.45 6.55
Jurkat 1.81 6.19
Jurkat (old) 0.96 7.04
Jurkat (mix) 1.39 6.62

Prepare Master Mix

X1.05 CHR22 CHR21 CHR8
2X iQ Super Mix 189 189 189
Primer F (3.3uM) 56.7 56.7 56.7
Primer R (3.3uM) 56.7 56.7 56.7
  • Aliquot 32ul to each well +8ul sample =40ul Total

Perform PCR reaction in real-time thermocycler

      Step1   96C, 3m
      Step2   95C, 30s
      Step3   62C, 1m
      Step4   72C, 1m
      Step5   Go to step2 repeat 39 times
      Step6   72C, 5m
      Step7   4C,  Forever
1 2
Jurkat (old) CV-iPS-F
Jurkat (mix)
Jurkat
NA12878
NA18507
NA20431
CV-Fibr
CV-iPS-B