Noi/NOTES/2014-9-10: Difference between revisions
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>Noi (Created page with "= MONOD V5 set A + CRC BSPP capture of tumor DNA samples and ctDNA from Illumina = * Conditions: ** Probe set: MONOPD V5 set A + CRC from Illumina ** Probe:target ratio: 200...") |
>Noi mNo edit summary |
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*** Set 2: BSPP capture on 4 bis-gDNA from colon primary tumor tissue and 4 WGBS libraries generated the matched pair colon cancer ctDNA. | *** 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: [[Noi/NOTES/2014-9-5| 2014-09-05:]], [[Noi/NOTES/2014-9-8| 2014-09-08]] | * Related notes: [[Noi/NOTES/2014-9-5| 2014-09-05:]], [[Noi/NOTES/2014-9-8| 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 == | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 | |||
{| {{table}} border = 1 | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Volume for 100ng''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''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 | |||
{| {{table}} border = 1 | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Volume for 100ng''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''Probe/Buffer Mix''' | |||
| align="center" style="width:70px;background:#f0f0f0;"|'''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 === | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 == | |||
* Did quick test PCR in 10l reaction | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 |
Latest revision as of 21:41, 14 September 2014
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