AlanFung:LabNotes/Capturing/2014-1-14: Difference between revisions
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==PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-4)== | ==PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-4)== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
| | | width="111" height="15" | Probes | ||
| width="77" | BSPP_GP1_V6 | | width="77" | BSPP_GP1_V6 | ||
| width="65" | illumina_155 | | width="65" | illumina_155 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
| align="center" height="15" | | | height="15" | Sample | ||
| align="center" | | | Jurkat_CT | ||
| align="center" | | | Jurkat_CT | ||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
| height="15" | Index | |||
| align="center" | 1 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
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| align="center" | | | align="center" | | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
| height="15" | | | height="15" | Probes | ||
| | | ppMALTA1 | ||
| | | ppMALTA1 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
| | | height="15" | Sample | ||
| | | Jurkat_gDNA | ||
| | | -ve control | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" |
Revision as of 00:36, 22 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.
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 with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-4)
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 |