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
Part I: 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 adjusted 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
- 6 cycles seemed to be over-amplified either based on gel image or qPCR curve (not shown). I will use the same PCR condition to amplify the rest of libraries with the same template concentration at 5 cycles.
Sample | Conc. (ng/ul) | Volume for 30ng (ul) | Sample | H2O to 80 ul |
NC-1 | 0.41 | 73.17 | NC-1 | 6.83 |
NC-2 | 1.39 | 21.58 | NC-2 | 58.42 |
NC-3 | 1.8 | 16.67 | NC-3 | 63.33 |
NC-5 | 0.47 | 63.83 | NC-5 | 16.17 |
NC-6 | 4.25 | 7.06 | NC-6 | 72.94 |
NC-7 | 0.47 | 63.83 | NC-7 | 16.17 |
NC-8 | 1.52 | 19.74 | NC-8 | 60.26 |
NC-9 | 3.72 | 8.06 | NC-9 | 71.94 |
NC-12 | 2.99 | 10.03 | NC-12 | 69.97 |
NC-13 | 1.64 | 18.29 | NC-13 | 61.71 |
NC-14 | 1.28 | 23.44 | NC-14 | 56.56 |
NC-15 | 1.29 | 23.26 | NC-15 | 56.74 |
NC-16 | 1.33 | 22.56 | NC-16 | 57.44 |
NC-17 | 3.86 | 7.77 | NC-17 | 72.23 |
NC-18 | 2.63 | 11.41 | NC-18 | 68.59 |
NC-19 | 0.47 | 63.83 | NC-19 | 16.17 |
NC-20 | 0.4 | 75.00 | NC-20 | 5.00 |
NC-21 | 0.41 | 73.17 | NC-21 | 6.83 |
NC-22 | 2.71 | 11.07 | NC-22 | 68.93 |
NC-23 | 1.76 | 17.05 | NC-23 | 62.95 |
NC-24 | 0.39 | 76.92 | NC-24 | 3.08 |
NC-27 | 6.88 | 4.36 | NC-27 | 75.64 |
NC-29 | 3.59 | 8.36 | NC-29 | 71.64 |
NC-30 | 6.46 | 4.64 | NC-30 | 75.36 |
PCR Mix
Components | Conc | unit | Final conc. | unit | Volume (ul) | 25rxn |
Purified 1st round DNA | 80.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 50.00 | 1,250.00 |
dNTP mix | 10 | mM | 0.25 | mM | 6.25 | 156.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 7.50 | 187.50 |
50X SYBR | 50 | X | 0.4 | X | 2.00 | 50.00 |
Phusion HF | 2 | unit/ul | 2.50 | 62.50 | ||
H2O | 101.75 | 2,543.75 | ||||
Total | 250.00 |
- - Aliquot 170ul to each tube + 80 ul of DNA template adjusted volume with H2O
- - Split ~60ul 4X in 96-well plate
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5 cycles --> 72C for 2min