Noi/NOTES/2014-11-4: Difference between revisions
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>Noi (Created page with "= SeqCap Epi probe pool (UMR v1) liquid hybridization on WGBS libraries generated from normal and cancer patients = '''Back to calendar''' [[Medi...") |
>Noi mNo edit summary |
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| Total||||||||||30.00|| | | Total||||||||||30.00|| | ||
|} | |} | ||
:- Aliquot 20ul + DNA template with | :- Aliquot 20ul + DNA template with adjusted H2O total 10ul | ||
:- Split 9ul 3X | :- Split 9ul 3X | ||
::98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5,6,7 cycles --> 72C for 2min | ::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 | :- Added 2ul of 6X loading dye | ||
:- Loaded 6ul of PCR and dye mix in 6% TBE gel | :- Loaded 6ul of PCR and dye mix in 6% TBE gel | ||
[[File:ZhangLab_2 2014-11-03 13hr 46min_test-reamp-wgbs.jpg| | [[File:ZhangLab_2 2014-11-03 13hr 46min_test-reamp-wgbs.jpg| 400px]] | ||
* 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. | |||
{| {{table}} border = 1 | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 30ng (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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''' | |||
{| {{table}} border =1 | |||
| align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 |
Revision as of 15:05, 8 November 2014
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 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