Revision as of 21:41, 14 September 2014 by >Noi
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