Jie:LabNotes/CpgSeq/2008-9-5: Difference between revisions
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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. | 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. | ||
==PCR(09/ | ==PCR(09/8/2008)== | ||
x5 | x5 |
Revision as of 17:29, 9 September 2008
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 3 tubes | 20ul | 0ul | 130ul | 17ng/ul x 30ul | 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 | see page 08-08-15 | 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 | see page 08-08-15 | 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.
PCR(09/8/2008)
x5 Template 10ul 2X iProof Mastermix 50ul 250ul AmpF6.2SoL (10uM) 4ul 20ul AmpR6.2SoL (10uM) 4ul 20ul 50X SYBG I 0.8ul 4ul H2O 31.2ul 156ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
File:20080907 captured PCR N0 4 5.jpg20080907_captured PCR_N0_4_5
File:20080907 captured PCR N0 6 14.jpg20080907_captured PCR_N0_6_14
File:20080907 captured PCR N0 15 Jurkat.jpg20080907_captured PCR_N0_15_Jurkat