AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4: Difference between revisions

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>Alan6017518
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*Aliquot 32ul to each well
*Aliquot 32ul to each well
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

Revision as of 18:39, 4 December 2009

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

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