Noi/NOTES/2014-12-16: Difference between revisions
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>Noi m (→Precedures) |
>Noi mNo edit summary |
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* Transfer 14.88ul of DNA to 95-well plate | * Transfer 14.88ul of DNA to 95-well plate | ||
** I re-organized the location of each sample on the well based on sample ID (small - large) | ** I re-organized the location of each sample on the well based on sample ID (small - large) | ||
* Aliquot 15.12ul of Probe & Buffer Mix in below table to each well | |||
* Total volume is 30ul. | |||
* Mix well, seal with plastic seal and spin down the plate | |||
* Layer with 30ul mineral oil | |||
* Seal with Aluminum seal and spin down the plate again. | |||
* Incubate the plate on thermocycler following program below | |||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | | align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | ||
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|} | |} | ||
* In 96-well plate, I re-organized the sample ID from small to large like from PCP2, PCP-3, PCP4,.... | * In 96-well plate, I re-organized the sample ID from small to large like from PCP2, PCP-3, PCP4,.... | ||
==== Probe & Buffer Mix ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1X''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''51X''' | |||
|- | |||
| MONOD V5A (LMS) 8.25ng/ul||12.12||618.18 | |||
|- | |||
| 10X Ampligase Buffer||3.00||153.00 | |||
|- | |||
| WGBS library (600ng)||14.88|| | |||
|- | |||
| Total||30.00|| | |||
|} | |||
'''Program''' | |||
-> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 23h | |||
-> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) | |||
--> '''No need to change program while adding PLN mix (keep the tube at 55C all the time)''' | |||
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 10C hold. | |||
--> '''Press proceed once''' before adding ExoI/III and waiting until temperature down to 37C before adding ExoI/III | |||
=== PLN (Polymerase/Ligase/ dNTP) mix solution Mix === |
Revision as of 05:17, 13 January 2015
LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient
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Continued from 2014-12-05
- Conditions:
- Probe set: MONOD V5 set A (LMS)
- Probe:target ratio: 150:1 for LMS probe set.
- Please note that I used very high amount of WGBS libraries for BSPP capture up to 600ng. This resulted in a very high amount of BSPP required for capture. I therefor reduced probe to target ratio from 200:1 to 125:1. The main reason is I do not have enough LMS BSPP to capture 50 samples with 600ng DNA each. For experiment I did for Illumina (Li Liu), I used 400ng of WGBS libraries, so I had enough probes to capture that amount. In order to be able to capture all unique molecule of ctDNA, I tried to increase WGBS libraries as much as possible. Ideally, Dr. Zhang wanted 1ug for capture.
- Sample:
- WGBS libraries of ctDNA from cancer patients prepared by KAPA Hyper Prep Kit 600ng.
- WGBS libraries of cfDNA from normal control (re-amplified) prepare by using RRBS protocol but skipping DNA digestion/fragmentation 600ng
Probe calculation
Probe set Probe size Probe:target ratio LMS 73,574 150:1 From 2014-12-08 exp., 400ng required 108ng (200:1). So 600ng (150:1) requires ~100ng of LMS probes
- Since the probe concentration a little low (8.25ng/ul), I will set up capture reaction in 300ul.
NOTE: WHAT ARE DIFFERENT FROM EXP. on 2014-12-08 (ILLUMINA, for Li Liu)
- Template 600ng instead of 400ng
- Probe: target ratio is 125:1 instead of 200:1
- Capture reaction 30ul instead of 25ul
Precedures
- I took 600ng of WGBS libraries in 1.5mL LoBind tube. Rationale: total volume of 600ng of WGBS libraries is too large to fit in 30ul capture reaction.
- Speedvac at 60C for ~20-30min to dry up DNA in the tube.
- Resuspend each tube with 14.88ul H2O which is the maximum volume of WGBS libraries to fit in 30ul capture reaction.
- Make sure that we resuspend DNA on the bottom of the tube very well.
- Transfer 14.88ul of DNA to 95-well plate
- I re-organized the location of each sample on the well based on sample ID (small - large)
- Aliquot 15.12ul of Probe & Buffer Mix in below table to each well
- Total volume is 30ul.
- Mix well, seal with plastic seal and spin down the plate
- Layer with 30ul mineral oil
- Seal with Aluminum seal and spin down the plate again.
- Incubate the plate on thermocycler following program below
Sample | Conc. (ng/ul) | Volume for 600ng (ul) |
NC-1 | 13.73 | 43.69 |
NC-2 | 16.00 | 37.50 |
NC-3 | 16.93 | 35.43 |
NC-5 | 13.07 | 45.92 |
NC-6 | 15.33 | 39.13 |
NC-7 | 13.73 | 43.69 |
NC-8 | 15.87 | 37.82 |
NC-9 | 16.53 | 36.29 |
NC-12 | 14.80 | 40.54 |
NC-13 | 13.19 | 45.50 |
NC-14 | 14.93 | 40.18 |
NC-15 | 13.60 | 44.12 |
NC-16 | 13.87 | 43.27 |
NC-17 | 13.47 | 44.55 |
NC-18 | 14.67 | 40.91 |
NC-19 | 13.47 | 44.55 |
NC-20 | 12.79 | 46.92 |
NC-21 | 12.31 | 48.75 |
NC-22 | 16.00 | 37.50 |
NC-23 | 13.60 | 44.12 |
NC-25 | 11.97 | 50.11 |
NC-27 | 14.67 | 40.91 |
NC-29 | 12.96 | 46.30 |
NC-30 | 15.47 | 38.79 |
Sample | Conc. (ng/ul) | Volume for 600ng (ul) |
PCP-9 | 26.13 | 22.96 |
PCP-4 | 21.73 | 27.61 |
PCP-8 | 19.07 | 31.47 |
PCP-6 | 20.13 | 29.80 |
PCP-7 | 22.80 | 26.32 |
PCP-2 | 22.13 | 27.11 |
PCP-3 | 21.47 | 27.95 |
PCP-5 | 21.47 | 27.95 |
6P-1 | 26.00 | 23.08 |
6P-8 | 24.80 | 24.19 |
6P-10 | 22.27 | 26.95 |
6P-9 | 20.27 | 29.61 |
6P-5 | 24.67 | 24.32 |
6P-2 | 19.60 | 30.61 |
6P-3 | 19.87 | 30.20 |
6P-4 | 19.20 | 31.25 |
7P-8 | 22.80 | 26.32 |
7P-3 | 19.07 | 31.47 |
7P-10 | 25.33 | 23.68 |
7P-7 | 29.60 | 20.27 |
7P-6 | 26.93 | 22.28 |
7P-1 | 14.67 | 40.91 |
7P-2 | 19.73 | 30.41 |
7P-5 | 18.00 | 33.33 |
- In 96-well plate, I re-organized the sample ID from small to large like from PCP2, PCP-3, PCP4,....
Probe & Buffer Mix
Components | 1X | 51X |
MONOD V5A (LMS) 8.25ng/ul | 12.12 | 618.18 |
10X Ampligase Buffer | 3.00 | 153.00 |
WGBS library (600ng) | 14.88 | |
Total | 30.00 |
Program -> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 23h -> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) --> No need to change program while adding PLN mix (keep the tube at 55C all the time) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 10C hold. --> Press proceed once before adding ExoI/III and waiting until temperature down to 37C before adding ExoI/III