Jie:LabNotes/CpgSeq/2008-10-10
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Digestion of PGP1F with Rnase I
PGP1F(35ng/ul) 100ul + 10U/ul Rnase I 2ul -> 37C 30mins -> 70C 20mins. IMR90(25ng/ul) 100ul + 10U/ul Rnase I 2ul -> 37C 30mins -> 70C 20min. Purify with Microcon column. Yield: IMR90 50ng/ul x 40ul. 260/280=0.96 PGPF1 57.8ng/ul x 50ul. 260/280=0.96
Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 | |
2 | BJ_iPS_11 | 140ng/ul x 2 tubes | 14.3ul | 5.7ul | 130ul | 101.9ng/ul x 20ul | 1.6/0.9 |
16 | IMB90_2 | 50ng/ul x 2 tubes | 20ul | 0ul | 130ul | 68ng/ul x 10ul | 1.48/0.93 |
17 | PGPF1 | 57.8ng/ul x 3 tubes | 20ul | 0ul | 130ul | 119.5ng/ul x 30ul | 1.79/1.28 |
- 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 | |
2 | BJ_iPS_11 | 101.9ng/ul x 20ul | 1ul | 4+1+1+5ul | 4+1.5+4.5 | 8+1.5+2.5 | 4+1.5+3.5 |
16 | IMB90_2 | 68ng/ul x 20ul | 1ul | 4+1+1+5ul | 4+1.5+4.5 | 8+1.5+2.5 | 4+1.5+3.5 |
17 | PGPF1 | 119.5ng/ul x 30ul | 1ul | 4+1+1+5ul | 4+1.5+4.5 | 8+1.5+2.5 | 4+1.5+3.5 |
bis_Jurkat | 200ng/ul | 1ul | 1+1+1+6ul | ||||
Jurkat | 200ng/ul | 1ul | cosmic probe 2ul | ||||
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.