Noi/NOTES/2014-12-8: Difference between revisions

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| CRC required (500:1)||3.76||ng
| CRC required (500:1)||3.76||ng
|}
'''Probe set Amount required(ng) Conc. (ng/ul) Volume/rxn'''
MONOD-V5A(LMS) 108.10                 12.10         8.93
CRC           3.76                 13.20         0.28
== BSPP capture set up ==
==== Probe Buffer Mix ====
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume'''
| align="center" style="width:80px;background:#f0f0f0;"|'''11.5 rxn mix'''
|-
| MONOD V5A (LMS) (12.1 ng/ul)||8.93||102.70
|-
| CRC (13.2ng/ul)||0.28||3.22
|-
| 10X Ampligase Buffer||2.50||28.75
|-
| H2O||1.29||14.84
|-
| Total||13.00||
|}
==== WGBS libraries ====
* We require 400ng of WGBS library for BSPP capture. The total volume of 400ng is quite large and exceed 25ul which is the total volume of BSPP capture. I simply pipette 400ng of WGBS libraries to 1.5mL LoBind tube. Then add H2O to bring total volume to 50ul.
{| {{table}} border = 1
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 70ul (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 400ul (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 50ul'''
|-
| KU_1||9.89||692.30||40.44||KU_1||9.56
|-
| KU_2||14.90||1,043.00||26.85||KU_2||23.15
|-
| KU_3||11.00||770.00||36.36||KU_3||13.64
|-
| KU_4||14.30||1,001.00||27.97||KU_4||22.03
|-
| KU_5||10.10||707.00||39.60||KU_5||10.40
|-
| KU_6||12.70||889.00||31.50||KU_6||18.50
|-
| KU_7||15.60||1,092.00||25.64||KU_7||24.36
|-
| KU_8||9.22||645.40||43.38||KU_8||6.62
|-
| KU_9||9.09||636.30||44.00||KU_9||6.00
|-
|-
| CRC conc. ||13.2||ng/ul
| KU_10||8.17||571.90||48.96||KU_10||1.04
|}
|}
* I evaporated WGBS libraries by closing the lid of the tube and made a hole by poking with needle size G22 (3 hole/tube) and evaporate at 60C for ~15min. The left over volume was between 5-12ul. I think to make it simple, I just dry up the whole tube then resuspend with equal volume of H2O to fit the capture volume. In this experiment I adjust total volume of WGBS library to 12ul with H2O
* I have no time to follow standard capture, so I reduce incubation time after adding KLN to 4h.
'''Program'''
-> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 10C hold.
* Note: After adding PLN, the incubation time could be varied between 2-20h.
=== PLN (Polymerase/Ligase/ dNTP) mix solution Mix ===

Revision as of 22:56, 10 December 2014

LMS + CRC BSPP capture on WGBS libraries prepared from ctDAN of CRC patient (Illumina)

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Continued from 2014-12-05

  • Conditions:
    • Probe set: MONOPD V5 set A (LMS) + CRC from Illumina
    • Probe:target ratio: 200:1 for LMS probe set, 500:1 for CRC probe set from Illumina
    • Sample: WGBS libraries prepared by KAPA Hyper Prep Kit (2014-12-05) amount 400ng

Probe calculation (Example)

Probe set 	        Probe size	Probe:target ratio
LMS                     73,574	              200:1
CRC	                 1,024	              500:1


Probe:target 200:1 '
Probe size 73574
Template 400 ng
Human gDNA MW 1.95E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (400ng) 2.05128E-19 mol (400ng/1.9500E+12 g/mol)
Probe required (200:1) 4.10256E-17 mol (2.5641E-20 * 200)
Probe MW (73,574 probes, 110nt) 2.636E+09 g/mol (73,574 probes * (110nt * 325Da/nt + 79Da))
Amount probe required 1.081E-07 g (2.051E-17mol * 2.636E+09g/mol)
Amount probe required 108 ng
CRC required (200:1) 1.50 ng
CRC required (500:1) 3.76 ng
Probe set	Amount required(ng)	Conc. (ng/ul)	Volume/rxn
MONOD-V5A(LMS)	108.10	                12.10	        8.93
CRC	          3.76	                13.20	        0.28

BSPP capture set up

Probe Buffer Mix

Components Volume 11.5 rxn mix
MONOD V5A (LMS) (12.1 ng/ul) 8.93 102.70
CRC (13.2ng/ul) 0.28 3.22
10X Ampligase Buffer 2.50 28.75
H2O 1.29 14.84
Total 13.00

WGBS libraries

  • We require 400ng of WGBS library for BSPP capture. The total volume of 400ng is quite large and exceed 25ul which is the total volume of BSPP capture. I simply pipette 400ng of WGBS libraries to 1.5mL LoBind tube. Then add H2O to bring total volume to 50ul.
Sample Conc. (ng/ul) Amount in 70ul (ng) Volume for 400ul (ul) Sample H2O to 50ul
KU_1 9.89 692.30 40.44 KU_1 9.56
KU_2 14.90 1,043.00 26.85 KU_2 23.15
KU_3 11.00 770.00 36.36 KU_3 13.64
KU_4 14.30 1,001.00 27.97 KU_4 22.03
KU_5 10.10 707.00 39.60 KU_5 10.40
KU_6 12.70 889.00 31.50 KU_6 18.50
KU_7 15.60 1,092.00 25.64 KU_7 24.36
KU_8 9.22 645.40 43.38 KU_8 6.62
KU_9 9.09 636.30 44.00 KU_9 6.00
KU_10 8.17 571.90 48.96 KU_10 1.04
  • I evaporated WGBS libraries by closing the lid of the tube and made a hole by poking with needle size G22 (3 hole/tube) and evaporate at 60C for ~15min. The left over volume was between 5-12ul. I think to make it simple, I just dry up the whole tube then resuspend with equal volume of H2O to fit the capture volume. In this experiment I adjust total volume of WGBS library to 12ul with H2O
  • I have no time to follow standard capture, so I reduce incubation time after adding KLN to 4h.
Program
-> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 10C hold.
  • Note: After adding PLN, the incubation time could be varied between 2-20h.

PLN (Polymerase/Ligase/ dNTP) mix solution Mix