Jie:LabNotes/CpgSeq/2009-5-20: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | | align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | ||
| align="center" style="background:#f0f0f0;"|'''template+cpg92k+H2O''' | | align="center" style="background:#f0f0f0;"|'''template+cpg92k+H2O''' | ||
| align="center" style="background:#f0f0f0;"|'''template+ | | align="center" style="background:#f0f0f0;"|'''template+control probes+H2O''' | ||
| align="center" style="background:#f0f0f0;"|'''reaction condition''' | | align="center" style="background:#f0f0f0;"|'''reaction condition''' | ||
|- | |- | ||
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|3||Jurkat||255.6ng/ul||1ul||1+5ul(#2_17ng/ul_05/20)+3ul||||55C anneal | |3||Jurkat||255.6ng/ul||1ul||1+5ul(#2_17ng/ul_05/20)+3ul||||55C anneal | ||
|- | |- | ||
|4||Jurkat||255.6ng/ul||1ul||||1+ | |4||Jurkat||255.6ng/ul||1ul||||1+1ulcpg30k,50ng/ul(06/13/08)+7ul|| | ||
|- | |- | ||
|5||Jurkat||142.8ng/ul||1ul||||1+1ul(cosmic,19.3ng/ul,04/29/08)+7ul|| | |5||Jurkat||142.8ng/ul||1ul||||1+1ul(cosmic,19.3ng/ul,04/29/08)+7ul|| |
Revision as of 18:34, 22 May 2009
bisulfite conversion of PGP samples
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | |
09_1 | NA21687*1 062507 | 241.1ng/ul x 1 tubes | 8ul | 12ul | 130ul | 95.4ng/ul x 10ul |
09_2 | NA21781*1 102307 | 240ng/ul x 1 tubes | 8ul | 12ul | 130ul | 120.2ng/ul x 10ul |
09_3 | NA21833*1 101807 | 253.9ng/ul x 1 tube | 8ul | 12ul | 130ul | 121.5ng/ul x 10ul |
09_4 | NA21660*1 053007 | 243.2ng/ul x 1 tube | 8ul | 12ul | 130ul | 120.3ng/ul x 10ul |
09_5 | NA21731*1 101807 | 217.4ng/ul x 1 tube | 10ul | 10ul | 130ul | 127.3ng/ul x 10ul |
09_6 | NA21846*1 100407 | 186.5ng/ul x 1 tube | 11ul | 9ul | 130ul | 151.3ng/ul x 10ul |
09_7 | NA21070*1 100406 | 329.7ng/ul x 1 tube | 6ul | 14ul | 130ul | 311.2ng/ul x 10ul |
Jurkat gDNA | 140ng/ul x 3 tube | 14ul | 6ul | 130ul | 255.6ng/ul x 20ul |
- Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
- Add 130ul of CT conversion reagent to samples.
- 98C 10min -> 64C 150min -> 4c overnight.
- Add 600ul M-Binding buffer to spin columns.
- Add the converted samples to the columns, close the gap and mix by inverting several times.
- Spin at 15,000rpm for 30sec.
- Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
- Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
- Add 200ul M-Wash Buffer, spin for 30 sec.
- Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
- Measure the DNA with Nanodrop:
RNA
set up the capture reaction
sample | sample concentration | 10xLigase buffer | template+cpg92k+H2O | template+control probes+H2O | reaction condition | |
1 | Jurkat | 255.6ng/ul | 1ul | 1+4ul(#1_22.3ng/ul_05/20)+4ul | 55C anneal | |
2 | Jurkat | 255.6ng/ul | 1ul | 1+4ul(#1_22.3ng/ul_05/20)+4ul | 60C anneal | |
3 | Jurkat | 255.6ng/ul | 1ul | 1+5ul(#2_17ng/ul_05/20)+3ul | 55C anneal | |
4 | Jurkat | 255.6ng/ul | 1ul | 1+1ulcpg30k,50ng/ul(06/13/08)+7ul | ||
5 | Jurkat | 142.8ng/ul | 1ul | 1+1ul(cosmic,19.3ng/ul,04/29/08)+7ul | ||
6 | 09_1 | 95.4ng/ul | 1ul | 2+5ul(#2_17ng/ul_05/20)+2ul | ||
7 | 09_7 | 311.2ng/ul | 1ul | 2+5ul(#2_17ng/ul_05/20)+2ul | ||
8 | negative control | 0 | 1ul | 0+5ul(#2_17ng/ul_05/20)+3ul |
dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.58ul 0.1ul 0.1ul 0.2ul (10mM)0.02ul x300 174ul 30ul 30ul 60ul 6ul
95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h ->
9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.