Noi/NOTES/2014-12-8: Difference between revisions
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>Noi mNo edit summary |
>Noi mNo edit summary |
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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 | |||
|- | |- | ||
| | | 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)
Back to calendar
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.