Noi/NOTES/2014-12-16: Difference between revisions

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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

PLN (Polymerase/Ligase/ dNTP) mix solution Mix