Jie:LabNotes/CpgSeq/2009-5-29
Bisulfite conversion of PGP DNA for cpg30k ASM validation[edit]
No | sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn |
20 | GM20431 | 256ng/ul x 1 tube | 8ul | 12ul | 130ul | 56.1ng/ul x 10ul |
21 | PGP1_iPS1 | 50ng/ul x 1 tubes | 10ul | 10ul | 130ul | 40.7ng/ul x 10ul |
22 | PGP1_L | 293ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 191.6ng/ul x 10ul |
23 | PGP3_L | 242ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 114.7ng/ul x 10ul |
24 | PGP9_L | 249ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 106.5ng/ul x 10ul |
09_8 | DF6-9-9_P16(4) | 504.8ng/ul x 3 tubes | 4ul | 16ul | 130ul | 285.7ng/ul x 10ul |
09_9 | foreskin | 583ng/ul x 2 tube | 4ul | 16ul | 130ul | 212.5ng/ul x 10ul |
- 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[edit]
No | sample | sample concentration | 10xLigase buffer | template+cpg30k(10ng/ul,2009-05-29 by Alan)+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 | template+Jan09#2(17ng/ul_05/20) vol+H2O |
09_8 | DF6-9-9_P16(4) | 285.7ng/ul x 10ul | 1ul | 1+3+1+3ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 | 2+5ul+2ul |
09_9 | foreskin | 212.5ng/ul x 10ul | 1ul | 1+3+1+3ul | 1+1.5+6.5 | 3+1.5+4.5 | 2+1.5+5.5 | 2+5ul+2ul |
positive control | Jurkat bis-gDNA 200ng/ul | 1ul | 1+3+1+6ul | |||||
negative control | 0 | 1ul | 0+3+1+6ul |
dNTP concentration 10mM(NEB)
ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x60 30ul 6ul 6ul 12ul 6ul
PCR with AmpF/R6.2Sol and iProof[edit]
x10 Template 5ul 2X iProof Mastermix 50ul 500ul AmpF6.2SoL (10uM) 4ul 40ul AmpR6.2SoL (10uM) 4ul 40ul 50X SYBG I 0.8ul 8ul H2O 36.2ul 362ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. I purified the amplicons with Qiaquick. Gel purified the cpg30k four subsets amplicons and normalized the final amplicons with 1:1 ratio. The 09_8 and 09_9 cpg97k amplicons concentrations are:
09_8: 11.3ng/ul x 30ul; 09_9: 13.7ng/ul x 30ul.
==PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40
I did the dUTP_PCR with template from the mixture captured targets, the Qiaquick purified captured targets of 97k, and the directed captured targets of 97k.
reaction system x8 x6 H2O 42.6ul 340.8ul 2x Master mix 50ul 400ul dUTP(1mM) 2ul 16ul AmpF6.3NH2(10uM) 2ul 16ul AmpR6.3NH2(10uM) 2ul 16ul 50x SYBG I 0.4ul 3.2ul template 0.25ul/each for cpg30k mixture, 0.05ul/each for purified cpg97k, 1ul/each for direct cpg97k. Total 100ul 1400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Qiaquick purification: No_09_8_30k: 34ng/ul x 60ul; No_09_8_97k: 21ng/ul x 60ul; No_09_9_30k: 36ng/ul x 60ul; No_09_8_97k: 24ng/ul x 60ul;
USER and S1 digestion[edit]
add 3ul USER to 30ul of each samples. 37C for 1h.
x4 10 x S1 nuclease buffer: 4 ul 16ul DNA after USER digestion: 33ul 33ul each S1 nuclease (10U/ul): 1ul 4ul ddH2O 2ul 8ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
end repair[edit]
positive control x5 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 12.5 dATP 2.5ul 2.5ul 12.5 10xendrepair buffer 2.5ul 2.5ul 12.5 enzyme 0.5ul 0.5ul 2.5 extra ATP(10mM) 2.5ul 2.5ul 12.5
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
Egel size selection[edit]
I did egel size selection for No09_8_30k, No09_9_30k and No09_9_97k. Select ~100bp fragments. The No09_8_97k goes to adapator ligation directly.
adapator ligation[edit]
x6 DNA 35ul 100uM Solexa_1 adaptor 0.5ul 3 100uM Solexa_2 adaptor 0.5ul 3 2xQuickLiage buffer 40ul 240 QuickLigase enzyme(NEB) 1ul 6 extra ATP(10mM) 4ul 24
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. egle size selection for No09_8_97k.
Nick-translation[edit]
set up the ligation system: x11 x5 Total 25ul 40 DNA 20ul 30 10x ThermoPol buffer 2.5ul 27.5ul 4 20 10mM dNTP 0.5ul 5.5ul 0.8 4 1mg/ml BSA 2.5ul 27.5ul 4 20 Bst polymerase(8U/ul) 1ul 11ul 1.5 7.5
65C for 25 minutes -> keep on ice.
do gel size selection for No09_8_30k, No09_9_30k and No09_9_97k, harvest DNA at ~160-175bp size range.
PCR of sequencig library[edit]
x11 x10 Nick-translated DNA 25ul Solexa_PCR_up(10uM) 2ul 22ul 200 Solexa_PCR_lo(10uM) 2ul 22ul 200 2xiProof master mix 50ul 550ul 500 50x SYBG 0.8ul 8.8ul 8 ddH2O 22ul 220ul 220 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
File:20090605 sol seq lib No09 8 9 97K.jpg20090605_sol seq lib No09_8_9_97K
Do qiaquick purification and egel cut. Gel quantification:
File:20090608 sol seq lib No09 8 9 97k quantification.jpg20090608_sol seq lib No09_8_9_97k quantification
No09_8_97k (no size selection before adaptor ligation): 1.5ng/ul (11nM) x 30ul No09_9_97k: 2ng/ul (15nM) x 15ul
Send the samples to BioGem:
DF6-9-9_P16(4): 11nM x 10ul x 2 lanes; foreskin: 15nM x 10ul x 1lane.
I resend th samples to Zhen Ye on 06/18/09 as follows:
Lane 3: DF6-9-9_P16(4)_97k_repeat: 11nM x 5ul. Lane 4: foreskin_97k_repeat: 7.5nM x 5ul.
I did the No09_8_30k and No09_9_30k solexa sequencing library at 06/04/09. refer to LabNotes on [1]