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
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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