Noi/NOTES/2014-9-10
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MONOD V5 set A + CRC BSPP capture of tumor DNA samples and ctDNA from Illumina[edit]
- Conditions:
- Probe set: MONOPD V5 set A + CRC from Illumina
- Probe:target ratio: 200:1
- Sample: bisDNA and WGBS libraires amount 100ng
- Sequencing libraries by BSPP capture will be included with some BSPP libraries generated on 2014-09-05.
- Set 1: BSPP capture on 4 WGBS libraries generated from cancer ctDNA from Illumina and 4 WGBS libraries generated from normal control cfDNA from Kang Zhang's lab.
- Set 2: BSPP capture on 4 bis-gDNA from colon primary tumor tissue and 4 WGBS libraries generated the matched pair colon cancer ctDNA.
- Related notes: 2014-09-05:, 2014-09-08
- I have made new batch of MONOD V5 set A probe: concentration = 20.9ng/ul.
- There are some samples that have a little low concentration. I need to take 100ul and evaporate to fit the capture volume of 20ul.
BSPP capture set up[edit]
Components | 1x rxn | 13.5x rxn |
MONOD V5 setA (20.9 ng/ul) | 1.29 | 17.44 |
CRC (1ng/ul) | 0.34 | 4.62 |
10X Ampligase Buffer | 2.00 | 27.00 |
Total | 3.63 |
- The maximum volume of DNA template is 20 - 3.63 = 16.37ul
Sample | Conc. (ng/ul) | Volume for 100ng | Need to evap? |
wNC-6 | 5.15 | 19.42 | YES |
wNC-9 | 3.95 | 25.32 | YES |
wNC-27 | 6.53 | 15.31 | |
wNC-30 | 7.48 | 13.37 | |
w6P-2 | 14.30 | 6.99 | |
w6P-3 | 5.77 | 17.33 | YES |
w6P-4 | 11.80 | 8.47 | |
w6P-5 | 6.45 | 15.50 | |
wcf1 | 8.33 | 12.00 | |
wcf2 | 5.07 | 19.72 | YES |
wcf3 | 9.43 | 10.60 | |
wcf4 | 9.60 | 10.42 | |
NTC | 0.00 | 0.00 |
- After evaporation for ~10min, each sample has different volume. I adjust volume to 16.37ul with H2O during setting up capture reaction.
- w = WGBS libraries
Sample | Conc. (ng/ul) | Volume for 100ng | H2O | Probe/Buffer Mix | Total |
wNC-6 | evap | 11.80 | 4.57 | 3.63 | 20.00 |
wNC-9 | evap | 16.37 | 0.00 | 3.63 | 20.00 |
wNC-27 | 6.53 | 15.31 | 1.06 | 3.63 | 20.00 |
wNC-30 | 7.48 | 13.37 | 3.00 | 3.63 | 20.00 |
w6P-2 | 14.30 | 6.99 | 9.38 | 3.63 | 20.00 |
w6P-3 | evap | 9.00 | 7.37 | 3.63 | 20.00 |
w6P-4 | 11.80 | 8.47 | 7.90 | 3.63 | 20.00 |
w6P-5 | 6.45 | 15.50 | 0.87 | 3.63 | 20.00 |
wcf1 | 8.33 | 12.00 | 4.37 | 3.63 | 20.00 |
wcf2 | evap | 11.30 | 5.07 | 3.63 | 20.00 |
wcf3 | 9.43 | 10.60 | 5.77 | 3.63 | 20.00 |
wcf4 | 9.60 | 10.42 | 5.95 | 3.63 | 20.00 |
NTC | 0.00 | 0.00 | 16.37 | 3.63 | 20.00 |
- 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 2ul KLN mix (2U/ul Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 4h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 10C hold.
KLN mix solution Mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (ul), total 30ul |
Hemo KlenTaq | 100 | % | 20 | % | 6.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 3.00 |
dNTP | 1 | mM | 100 | uM | 3.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 3.00 |
H2O | 15.00 |
Amplification[edit]
- Did quick test PCR in 10l reaction
Components | 1x rxn | 14 rxn mix |
Captured template | 1.50 | 0.00 |
10uM AmpF6.4Sol | 0.20 | 2.80 |
10uM AmpR6.3.IndX | 0.20 | 2.80 |
2X KAPA MM | 5.00 | 70.00 |
H2O | 3.10 | 43.40 |
Total | 10.00 |
Program (Eppendorf Realplex) 95C 30s -> (95C 10s -> 58C 20s -> 72C 20s)x8 -> (95C 10s -> 72C 20s)xXX -> 72C 3min