Jie:LabNotes/CpgSeq/2009-5-20: 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;"|''' '''
| 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+cpg92k+H2O'''
| align="center" style="background:#f0f0f0;"|'''template+cpg30k+H2O'''
| align="center" style="background:#f0f0f0;"|'''reaction condition'''
| align="center" style="background:#f0f0f0;"|'''template+v2.3(20ng/ul) vol+H2O'''
|-
|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+5ul(#2_17ng/ul_05/20)+3ul||
|-
|5||Jurkat||  ||
|-
|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 -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.

Revision as of 18:18, 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+cpg30k+H2O reaction condition template+v2.3(20ng/ul) vol+H2O
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+5ul(#2_17ng/ul_05/20)+3ul
5 Jurkat
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.