Noi/NOTES/2014-11-4: Difference between revisions

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(Created page with "= SeqCap Epi probe pool (UMR v1) liquid hybridization on WGBS libraries generated from normal and cancer patients = '''Back to calendar''' [[Medi...")
 
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| Total||||||||||30.00||
| Total||||||||||30.00||
|}
|}
:- Aliquot 20ul + DNA template with adjustable H2O total 10ul
:- 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| 500px]]
  [[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