Noi/NOTES/2014-11-4

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SeqCap Epi probe pool (UMR v1) liquid hybridization on WGBS libraries generated from normal and cancer patients

Back to calendar SeqCap Epi User Guide

  • Since the kit we purchased contains only SeqCap EZ HE-Oligo Kit A used to block the adaptor region also barcode adaptor of WGBS, I will pool only libraries that have the match barcode in set A

SeqCap EZ HE-Oligo Kit A oligo list

-HE Universal Oligo 1
-HE Index 2 Oligo
-HE Index 4 Oligo
-HE Index 5 Oligo
-HE Index 6 Oligo
-HE Index 7 Oligo
-HE Index 12 Oligo
-HE Index 13 Oligo
-HE Index 14 Oligo
-HE Index 15 Oligo
-HE Index 16 Oligo
-HE Index 18 Oligo
-HE Index 19 Oligo

Re-amplification of WGBS libraries of normal plasma samples

  • Most of normal plasma WGBS libraries have very low yield. I need to re-amplify to increase the yield and have some left for future experiment.
  • I did a test on few samples, including NC-7, NC-7, and NC-30, for number of cycle to re-amplify .

Template amount for 30ul run

Sample Conc. (ng/ul) Volume for 3.6ng H2O to 10ul
NC-7 0.47 7.66 2.34
NC-8 1.52 2.37 7.63
NC-30 6.46 0.56 9.44

PCR Mix

Components Conc unit Final conc. unit Volume (ul) 3.5 rxn mix
Purified 1st round DNA 10.00 0.00
5X Phusion HF buffer 5 X 1 X 6.00 21.00
dNTP mix 10 mM 0.25 mM 0.75 2.63
TruS_F/R 10 uM 0.3 uM 0.90 3.15
50X SYBR 50 X 0.4 X 0.24 0.84
Phusion HF 2 unit/ul 0.30 1.05
H2O 11.81 41.34
Total 30.00
- Aliquot 20ul + DNA template with adjustable H2O total 10ul
- Split 9ul 3X
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5,6,7 cycles --> 72C for 2min
- Added 2ul of 6X loading dye
- Loaded 6ul of PCR and dye mix in 6% TBE gel
File:ZhangLab 2 2014-11-03 13hr 46min test-reamp-wgbs.jpg