Jie:LabNotes/CpgSeq/2009-1-22
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6th Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit
No | sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 |
21 | PGP1_iPS1 | 50ng/ul x 3 tubes | 20ul | 0ul | 130ul | 179.1ng/ul x 10ul | 1.97/1.72 |
22 | PGP1_L | 293ng/ul x 3 tubes | 6.8ul | 13.2ul | 130ul | 368.7ng/ul x 20ul | 2.13/2.04 |
23 | PGP3_L | 242ng/ul x 3 tubes | 6.8ul | 13.2ul | 130ul | 238.6ng/ul x 20ul | 2.07/1.89 |
24 | PGP9_L | 249ng/ul x 3 tubes | 6.8ul | 13.2ul | 130ul | 223.7ng/ul x 20ul | 2.05/1.89 |
25 | PGP1F.2 | 25ng/ul x 4 tubes | 20ul | 0ul | 130ul | 84ng/ul x 10ul | 1.67/1.2 |
26 | PGP9F.2 | 14ng/ul x 4 tube | 20ul | 0ul | 130ul | 35.1ng/ul x 10ul | 1.2/0.66 |
- 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
No | 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 |
21 | PGP1_iPS1 | 179.1ng/ul x 10ul | 1ul | 1+1+1+6ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 |
22 | PGP1_L | 368.7ng/ul x 10ul | 1ul | 1+1+1+6ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 |
23 | PGP3_L | 238.6ng/ul x 10ul | 1ul | 1+1+1+6ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 |
24 | PGP9_L | 223.7ng/ul x 10ul | 1ul | 1+1+1+6ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 |
25 | PGP1F.2 | 84ng/ul x 10ul | 1ul | 7+1+1+0ul | |||
26 | PGP9F.2 | 35.1ng/ul x 10ul | 1ul | 7+1+1+0ul | |||
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 |
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.
PCR(08/16/2008)
x8 Template 10ul 2X iProof Mastermix 50ul 400ul AmpF6.2SoL (10uM) 4ul 32ul AmpR6.2SoL (10uM) 4ul 32ul 50X SYBG I 0.8ul 6.4ul H2O 31.2ul 249.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.