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

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  Gel 3
  Gel 3
  Ladder Jurkat(22) Jurkat(21) Jurkat(8) Jurkat_Old(22) Jurkat_Old(21) Jurkat_Old(8) Jurkat_Mix(22) Jurkat_Mix(21) Jurkat_Mix(8)
  Ladder Jurkat(22) Jurkat(21) Jurkat(8) Jurkat_Old(22) Jurkat_Old(21) Jurkat_Old(8) Jurkat_Mix(22) Jurkat_Mix(21) Jurkat_Mix(8)
15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
15ul h2o+3ul 6X loading dye+2ul sample
Run at 250V for 20min

Revision as of 00:30, 5 December 2009

qPCR Validation(3 Bisulfite Conversion 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 19.85

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

CHR22 CHR21 CHR8
NA12878 NA12878 NA12878 Jurkat (mix) CHR22
NA18507 NA18507 NA18507 Jurkat (mix) CHR21
NA20431 NA20431 NA20431 Jurkat (mix) CHR8
CV-Fibr CV-Fibr CV-Fibr
CV-iPS-B CV-iPS-B CV-iPS-B
CV-iPS-F CV-iPS-F CV-iPS-F
Jurkat Jurkat Jurkat
Jurkat (old) Jurkat (old) Jurkat (old)
      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

TBE Gel Verification

  • Run 3 10 well gels
Gel 1
Ladder NA12878(22) NA12878(21) NA12878(8) NA18507(22) NA18507(21) NA18507(8) NA20431(22) NA20431(21) NA20431(8)
Gel 2
Ladder CV-Fibr(22) CV-Fibr(21) CV-Fibr(8) CV-iPSB(22) CV-iPSB(21) CV-iPSB(8) CV-iPSF(22) CV-iPSF(21) CV-iPSF(8) 
Gel 3
Ladder Jurkat(22) Jurkat(21) Jurkat(8) Jurkat_Old(22) Jurkat_Old(21) Jurkat_Old(8) Jurkat_Mix(22) Jurkat_Mix(21) Jurkat_Mix(8)
15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
15ul h2o+3ul 6X loading dye+2ul sample 
Run at 250V for 20min