AlanFung:LabNotes/Capturing/2014-1-14: Difference between revisions
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*Library:13ul library + 13ul 6x loading dye | *Library:13ul library + 13ul 6x loading dye | ||
*One well in a 5 well TBE gel, run at 200V for 30 mins | *One well in a 5 well TBE gel, run at 200V for 30 mins | ||
==Send library to illumina for sequencing== | |||
===Prepare sequencing primer=== | |||
*Read 1 sequencing primer: | |||
*Index Read: | |||
*Read 2 sequencing primer: | |||
*Dilute stock (100uM) primer to 0.5uM using HT1 buffer | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
| align="center" width="116" height="16" | | |||
|style="font-weight:bold" width="263" | Stock Primer 100uM | |||
|style="font-weight:bold" width="179" | HT1 | |||
|style="font-weight:bold" width="156" | Total Volume | |||
|style="font-weight:bold" width="179" | Final Concentration (uM) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="16" | Volume (ul) | |||
| align="center" | 3 | |||
| align="center" | 597 | |||
| align="center" | 600 | |||
| align="center" | 0.5 | |||
|} | |||
Library concentration (qPCR), we normally load it at pM |
Revision as of 22:53, 28 January 2014
Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set
- probes prep notes LabNotes/Probe/2014-1-7
[[1]]
Capturing Jurkat gDNA w ppMALT1 ordered form IDT
Bisulfite Conversion of Jurkat gDNA
- Bisulfite conversion with zymo methylation gold
- Convert 500ng of Jurkat gDNA (2 rxn, 100ng total)
- Add 130ul of lighting conversion reagent to 5ul of jurkat gDNA and 15ul of ddH2O
- Mix and centrifuge
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
- Add 600ul M-binding buffer to column
- load the sample into column, mix by inverting column and centrifuge at 14000rpm for 30sec
- discard flow through
- add 100ul m-wash buffer to the column and centrifuge at 14000rpm for 30sec
- add 200ul L-Desulphonation buffer to the column and sit at RT for 20 minutes and centrifuge at 14000rpm for 30sec
- add 200ul of m-wash buffer to the column and centrifuge at 14000rpm for 30sec
- Add 10ul of EBuffer to column sit for 5 mintues and centrifuge at 14000rpm for 30sec (2 rxn total)
ssDNA Qubit assay
- 47.1ng/ul *20ul = 942ng
- Stored in Maytag -20C (Alan Misc. #9)
Probe calculations
illumina methylation seq pool 155
ppMALAT1
GP1_V6
Capture setup
GP1_V6 | ||||||
Sample | Conc. (ng/ul) | Volume for 200ng (ul) | H2O | GP1_V6 | 10X ampligase Buffer | Total |
CT Jurkat | 47.1 | 4.25 | 11.47 | 2.28 | 2 | 20 |
illumina methylation seq pool | ||||||
Sample | Conc. (ng/ul) | Volume for 200ng (ul) | H2O | illumina methylation seq pool | 10X ampligase Buffer | Total |
CT Jurkat | 47.1 | 4.25 | 10.38 | 3.37 | 2.00 | 20 |
ppMALAT1 | ||||||
Sample | Conc. (ng/ul) | Volume for 200ng (ul) | H2O | ppMALAT1 | 10X ampligase Buffer | Total |
Jurkat | 100 | 2 | 15.34 | 0.66 | 2.00 | 20 |
Negative Control | n/a | n/a | 17.34 | 0.66 | 2.00 | 20 |
illumina methylation seq pool 155 probes setup
Program stored under Alan/aCPG -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 58C 20h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
GP1_V6 & ppMALAT1 setup
Program stored under Kun/CPG -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- GP1_V6-Jurkat Stored in Maytag -20C (Alan Misc. #24)
- illumina_155-Jurkat Stored in Maytag -20C (Alan Misc. #25, remove for amplification done on 01282014)
- ppMALTA1-Jurkat Stored in Maytag -20C (Alan Misc. #26)
- ppMALTA1-H2O Stored in Maytag -20C (Alan Misc. #27)
Prepare SLN Mix
SLN Mix | |||||
Components | Stock Concentration | Unit | Final Concentration | Unit | Prepare Volume (ul) |
AmpliTaq Stoffel | 10 | U/ul | 2 | U/ul | 2.6 |
Ampligase | 5 | U/ul | 0.5 | U/ul | 1.3 |
dNTP | 1 | mM | 100 | uM | 1.3 |
10X Ampligase Buffer | 10 | X | 1 | X | 1.3 |
H2O | n/a | n/a | n/a | n/a | 6.5 |
Total | n/a | n/a | n/a | n/a | 13 |
Prepare Exo Mix
- Mix 1:1 Exo I & Exo III (9ul total)
PCR Amplification
(AmpF6.4Sol and AmpR6.3 for GP1_V6 & illumina_155)
(ISB_CA_AF and ISB_CA_AR_T2 for ppMALTA1) stored in #18, #19 Alan Misc. -20C
Probes | BSPP_GP1_V6 | illumina_155 |
Sample | Jurkat_CT | Jurkat_CT |
Index | 1 | 2 |
Components | 1x rxn | 1x rxn |
Captured Template | 20 | 20 |
10uM AmpF6.4Sol | 1 | 1 |
10uM AmpR6.3Indx | 1 | 1 |
2X KAPA SYBR MM | 25 | 25 |
H2O | 3 | 3 |
Probes | ppMALTA1 | ppMALTA1 |
Sample | Jurkat_gDNA | -ve control |
Components | 1x rxn | 1x rxn |
Captured Template | 20 | 20 |
10uM ISB_CA_AF | 1 | 1 |
10uM ISB_CA_AR_T2 | 1 | 1 |
2X KAPA SYBR MM | 25 | 25 |
H2O | 3 | 3 |
Program (Eppendorf Realplex stored under Alan/ampF6.4_template) 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- After the 8+15 cycle only GP1_V6 captured seq library got amplified
- After meeting with Dr. Zhang, he suggests adding more cycles to the remaining 3 captured product
- Amplification happened immediately for all 3 captures, that includes the negative control, which makes me think it is unspecific amplification. I will run a PAGE gel to verify the amplicon size
- GP1_V6 amplicon Stored in Maytag -20C (Alan Misc. #20)
- illumina_155 amplicon Stored in Maytag -20C (Alan Misc. #21, removed for gel size selection)
- ppMALTA1_Jurkat amplicon Stored in Maytag -20C (Alan Misc. #22)
- ppMALTA1_H2O amplicon Stored in Maytag -20C (Alan Misc. #23)
PAGE Gel
File:ZhangLab 2 2014-01-21 13hr 54min.jpg
- LM-GP1_V6-illumina_155-ppMALAT1-ppMALAT1_Negative
- illumina_155: probe:99nt, gap:99nt, primer: Dr. Zhang agrees that the amplicon at around 300bp should be the captured product. We will perform a size selection and send it to illumina for miseq run
Perform PAGE Size Selection on illumina_155pp-CT_Jurkat
- Purify amplicon using minelute, elute with 12ul EB buffer
- nanodrop result 37.9ng/ul * 12ul = 454.8ng/ul
- PAGE
Re-amplify illumina_JurkatCT 01/28/2014
Probes | illumina_155 |
Sample | Jurkat_CT |
Index | 1 |
Captured Template | 4 |
10uM AmpF6.4Sol | 1 |
10uM AmpR6.3Ind1 | 1 |
2X KAPA SYBR MM | 25 |
H2O | 19 |
Total | 50 |
Program (Eppendorf Realplex stored under Alan/ampF6.4_template) 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x30 -> 72C 3min
- Minelute with 15ul EB Buffer
- 14.3ng/ul *15ul = 214.5ng
PAGE Size Selection
- Lowmass-Ladder: 1ul + 12ul h2o + 13ul loading dye
- Library:13ul library + 13ul 6x loading dye
- One well in a 5 well TBE gel, run at 200V for 30 mins
Send library to illumina for sequencing
Prepare sequencing primer
- Read 1 sequencing primer:
- Index Read:
- Read 2 sequencing primer:
- Dilute stock (100uM) primer to 0.5uM using HT1 buffer
Stock Primer 100uM | HT1 | Total Volume | Final Concentration (uM) | |
Volume (ul) | 3 | 597 | 600 | 0.5 |
Library concentration (qPCR), we normally load it at pM