AlanFung:LabNotes/Capturing/BisFirstExon 2010 1 7

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Revision as of 01:00, 8 January 2010 by >Alan6017518 (→‎Size Selection)
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Qubit Quantification (ssDNA)[edit]

  • Prepare 4 Qubit Assay Tubes (2 for ladder, 2 for samples)
  • Need 800ul Diluted Dye
Mix 4ul dye in 796 Quant -iT buffer
  • Mix 10ul standard with 190 ul working soln.
  • Mix 2ul sample with 198ul working soln.
  • Vortex all tubes for 2-3 secs
  • Incubate for 2 mins at RT

Result[edit]

CT Converted Jurkat 1.1ng/ul * 20ul = 22ng
CT Converted CV-Fibr 16.2ng/ul * 20ul = 324ng
Start (ng) End (ng) Yield %
CV-Fibr 500 324 64.8
Jurkat 100 22 22
Yield is lower than expected, Jurkat DNA might be degraded already.

qPCR Validation(3 Bisulfite Conversion Primers)[edit]

  • Start with 10ng template for each set of primer (30ng Total)
Concentration Volume Make up to 24ul
CV-Fibr 16.2 1.85 22.15
Jurkat 1.1 10 14

Prepare SuperMix[edit]

  • Prepare 3 tubes for 3 different primers mix
CHR22 CHR21 Chr8
2X iQ Super Mix 44 44 44
Primer F (3.3uM) 13.2 13.2 13.2
Primer R (3.3uM) 13.2 13.2 13.2
  • Aliquot 32ul to each well +8ul sample =40ul Total

Perform PCR reaction in real-time thermocycler

Setup
CV-Fibr (CHR22)
CV-Fibr (CHR21)
CV-Fibr (CHR8)
Jurkat (CHR22)
Jurkat (CHR21)
Jurkat (CHR8)
      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
8ul h2o+1.5ul 6x loading dye+0.5ul 25bp ladder
6.5ul h2o+1.5ul 6X loading dye+2ul sample 
Run at 250V for 20min
Lane Setup
1 LM Ladder
2 CV-Fibr (CHR22)
3 CV-Fibr (CHR21)
4 CV-Fibr (CHR8)
5 Jurkat (CHR22)
6 Jurkat (CHR21)
7 Jurkat (CHR8)
8 NA12878 PCR_AMP F/R 6.3r Sol
9 NA18507 PCR_AMP F/R 6.3r Sol
10 NA20431 PCR_AMP F/R 6.3r Sol
11 Jurkat_New PCR_AMP F/R 6.3r Sol
12 Jurkat _Old PCR_AMP F/R 6.3r Sol

File:ZhangLab 2 2010-01-07 14hr 55min.jpg

  • CT Conversion Works
  • PCR with sequencing adapters works on NA20431, mix NA20431 (6.3 & 6.3r Sol) and perform size selection

Qiaquick[edit]

  • Mix the PCR amplified products of NA20431 together
  • Purify with 2X Qiaquick columns, eluted each with 22 ul EB.
  • Measure concentration with nanodrop
Result: 64.4ng/ul * 44ul = 2833.6ng

Size Selection[edit]

  • For optimal results load up to 700ng of total sample per well (2833.6/700=Run 4 wells)
  • Fill any unused well with 30ul EB Buffer
  • Mix 15ul EB buffer with 0.5ul LM bp ladder in a 0.2ml tube
  • Load 20ul end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
  • Run the SizeSelect program for 12~14 min. Pause when the band just move into the middle collection well
  • Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
  • Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
  • Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.

File:ZhangLab 2 2010-01-07 16hr 53min.jpg