Noi/NOTES/2014-12-16: Difference between revisions
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>Noi (Created page with "= LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient = '''Back to calendar'''<br> [[Noi/NOTE...") |
>Noi mNo edit summary |
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* Conditions: | * Conditions: | ||
** Probe set: MONOD V5 set A (LMS) | ** Probe set: MONOD V5 set A (LMS) | ||
** Probe:target ratio: 150:1 for LMS probe set. Please note that | ** Probe:target ratio: 150:1 for LMS probe set. | ||
** Sample: WGBS libraries prepared by KAPA Hyper Prep Kit | ***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. | ||
==== Probe calculation | ** 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''' | '''Probe set Probe size Probe:target ratio''' | ||
LMS 73,574 150:1 | 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 |
Revision as of 04:46, 13 January 2015
LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient
Back to calendar
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