AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4

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qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)

  • Start with 10ng template for each set of primer (30ng Total)
Sample Sample Concentration ng/ul To get 30ng 1/10 Dilution To get 30ng
NA12878 17.6 1.7
NA18507 12.4 2.42
NA20431 50.6 0.59
CV-Fibr 4.84 6.2
CV-iPS-B 7.27 4.13
CV-iPS-F 6.91 4.34
Jurkat 55.4 0.54 5.54 5.42
Jurkat (old) 104 0.29 10.4 2.88
  • Mix 50/50 old/new Jurkat to make up 30ng
New: 2.71ul
Old: 1.44ul
Sample Volume Make it up to 24ul
NA12878 1.7 22.3
NA18507 2.42 21.58
NA20431 0.59 23.41
CV-Fibr 6.2 17.8
CV-iPS-B 4.13 19.87
CV-iPS-F 4.34 19.66
Jurkat 5.42 18.58
Jurkat (old) 2.88 21.12
Jurkat (mix) (NEW)2.71+(OLD)1.44 24

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