Jie:LabNotes/CpgSeq/2009-5-29: Difference between revisions

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*Measure the DNA with Nanodrop:
*Measure the DNA with Nanodrop:
RNA
RNA
==set up the capture reaction==
{| border="1" cellpadding="5" cellspacing="0" align="center"
|-
| align="center" style="background:#f0f0f0;"|'''No '''
| align="center" style="background:#f0f0f0;"|'''sample '''
|align="center" style="background:#f0f0f0;"|'''sample concentration'''
| align="center" style="background:#f0f0f0;"|'''10xLigase buffer'''
| align="center" style="background:#f0f0f0;"|'''template+cpg30k(10ng/ul,2009-05-29 by Alan)+suppressor(8nM each)vol+H2O'''
| align="center" style="background:#f0f0f0;"|'''template+v2.1(21ng/ul) vol+H2O'''
| align="center" style="background:#f0f0f0;"|'''template+v2.2(21ng/ul) vol+H2O'''
| align="center" style="background:#f0f0f0;"|'''template+v2.3(20ng/ul) vol+H2O'''
| align="center" style="background:#f0f0f0;"|'''template+Jan09#2 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+
|-
|09_9||foreskin||212.5ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
| ||positive control||200ng/ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
| ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|}

Revision as of 22:04, 29 May 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 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+
09_9 foreskin 212.5ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
positive control 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
negative control 0 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5