Jie:LabNotes/CpgSeq/2009-5-29: Difference between revisions
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==adapator ligation== | ==adapator ligation== | ||
positive control negative control x6 | |||
DNA | DNA 35ul 13ul ddH2O 13ul | ||
100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul | 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 3 | ||
100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul | 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 3 | ||
2xQuickLiage buffer | 2xQuickLiage buffer 20ul 15ul 15ul 120 | ||
QuickLigase enzyme(NEB) 1ul 1ul 1ul | QuickLigase enzyme(NEB) 1ul 1ul 1ul 6 | ||
extra ATP(10mM) | extra ATP(10mM) 4ul 2.5ul 2.5ul 24 | ||
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. | Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. | ||
TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O. | TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O. |
Revision as of 18:08, 4 June 2009
Bisulfite conversion of PGP DNA for cpg30k ASM validation
No | sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn |
20 | GM20431 | 256ng/ul x 1 tube | 8ul | 12ul | 130ul | 56.1ng/ul x 10ul |
21 | PGP1_iPS1 | 50ng/ul x 1 tubes | 10ul | 10ul | 130ul | 40.7ng/ul x 10ul |
22 | PGP1_L | 293ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 191.6ng/ul x 10ul |
23 | PGP3_L | 242ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 114.7ng/ul x 10ul |
24 | PGP9_L | 249ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 106.5ng/ul x 10ul |
09_8 | DF6-9-9_P16(4) | 504.8ng/ul x 3 tubes | 4ul | 16ul | 130ul | 285.7ng/ul x 10ul |
09_9 | foreskin | 583ng/ul x 2 tube | 4ul | 16ul | 130ul | 212.5ng/ul x 10ul |
- 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
No | sample | sample concentration | 10xLigase buffer | template+cpg30k(10ng/ul,2009-05-29 by Alan)+suppressor(8nM each)vol+H2O | template+v2.1(21ng/ul) vol+H2O | template+v2.2(21ng/ul) vol+H2O | template+v2.3(20ng/ul) vol+H2O | template+Jan09#2(17ng/ul_05/20) vol+H2O |
09_8 | DF6-9-9_P16(4) | 285.7ng/ul x 10ul | 1ul | 1+3+1+3ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 | 2+5ul+2ul |
09_9 | foreskin | 212.5ng/ul x 10ul | 1ul | 1+3+1+3ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 | 2+5ul+2ul |
positive control | Jurkat bis-gDNA 200ng/ul | 1ul | 1+3+1+6ul | |||||
negative control | 0 | 1ul | 0+3+1+6ul |
dNTP concentration 10mM(NEB)
ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x60 30ul 6ul 6ul 12ul 6ul
PCR with AmpF/R6.2Sol and iProof
x10 Template 5ul 2X iProof Mastermix 50ul 500ul AmpF6.2SoL (10uM) 4ul 40ul AmpR6.2SoL (10uM) 4ul 40ul 50X SYBG I 0.8ul 8ul H2O 36.2ul 362ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. I purified the amplicons with Qiaquick. Gel purified the cpg30k four subsets amplicons and normalized the final amplicons with 1:1 ratio. The 09_8 and 09_9 cpg97k amplicons concentrations are:
09_8: 11.3ng/ul x 30ul; 09_9: 13.7ng/ul x 30ul.
==PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40
I did the dUTP_PCR with template from the mixture captured targets, the Qiaquick purified captured targets of 97k, and the directed captured targets of 97k.
reaction system x8 x6 H2O 42.6ul 340.8ul 2x Master mix 50ul 400ul dUTP(1mM) 2ul 16ul AmpF6.3NH2(10uM) 2ul 16ul AmpR6.3NH2(10uM) 2ul 16ul 50x SYBG I 0.4ul 3.2ul template 0.25ul/each for cpg30k mixture, 0.05ul/each for purified cpg97k, 1ul/each for direct cpg97k. Total 100ul 1400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Qiaquick purification: No_09_8_30k: 34ng/ul x 60ul; No_09_8_97k: 21ng/ul x 60ul; No_09_9_30k: 36ng/ul x 60ul; No_09_8_97k: 24ng/ul x 60ul;
USER and S1 digestion
add 3ul USER to 30ul of each samples. 37C for 1h.
x4 10 x S1 nuclease buffer: 4 ul 16ul DNA after USER digestion: 33ul 33ul each S1 nuclease (10U/ul): 1ul 4ul ddH2O 2ul 8ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
end repair
positive control x5 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 12.5 dATP 2.5ul 2.5ul 12.5 10xendrepair buffer 2.5ul 2.5ul 12.5 enzyme 0.5ul 0.5ul 2.5 extra ATP(10mM) 2.5ul 2.5ul 12.5
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
Egel size selection
adapator ligation
positive control negative control x6 DNA 35ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 3 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 3 2xQuickLiage buffer 20ul 15ul 15ul 120 QuickLigase enzyme(NEB) 1ul 1ul 1ul 6 extra ATP(10mM) 4ul 2.5ul 2.5ul 24
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.