Jie:LabNotes/CpgSeq/2008-9-5

From ZhangLabWiki
Revision as of 22:27, 5 September 2008 by >Jie deng
Jump to navigation Jump to search

Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit (3rd)

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
4 hFib2 68.4ng/ul x 3 tubes 20ul 0ul 130ul 34.1ng/ul x 30ul 2.1/2.7
5 hFib2_iPS4 51.9ng/ul x 3 tubes 20ul 0ul 130ul 47.4ng/ul x 30ul 2.0/2.5
6 IMB90 50ng/ul x 1 tube 20ul 0ul 130ul 36ng/ul x 10ul 1.8/2.4
14 Hues66 99ng/ul x 4 tubes 20ul 0ul 130ul 17ng/ul x 40ul 1.8/1.9
15 Hues63 207ng/ul x 3 tubes 10ul 10ul 130ul 132ng/ul x 30ul 2.2/2.7


  • 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 system

sample sample concentration 10xLigase buffer template+cpg30k(50.7ng/ul)+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
4 hFib2 34.1ng/ul x 30ul + 28.4ng/ul x 20ul 1ul 6+1+1+1ul 8+1.5 10+1.5 x 2 tubes 10+1.5
5 hFib2_iPS4 47.4ng/ul x 30ul + 37.8ng/ul x 20ul 1ul 6+1+1+1ul 8+1.5 10+1.5 x 2 tubes 10+1.5
6 IMB90 36ng/ul x 10ul + 22.9ng/ul x 20ul 1ul 8+1.5 10+1.5 x 2 tubes 10+1.5
14 Hues66 17ng/ul x 40ul 1ul 8+1+1 10+1.5 12+1.5 10+1.5
15 Hues63 132ng/ul x 30ul 1ul 4+1+1+3 6+1.5+1.5 10+1.5 8+1.5
Jurkat 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 1.5+1.5+6
negative control 0 1ul 1+1+1+6ul 1+1.5+6.5 2+1.5+5.5 1.5+1.5+6


 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.