Jie:LabNotes/CpgSeq/2009-5-29
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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
==PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40
reaction system x14 x2 H2O 42.6ul 596.4ul 2x Master mix 50ul 700ul dUTP(1mM) 2ul 28ul AmpF6.3NH2(10uM) 2ul 28ul AmpR6.3NH2(10uM) 2ul 28ul 50x SYBG I 0.4ul 5.6ul template(4ng/ul) 0.5ul 2 uleach Total 100ul 1400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C