Jie:LabNotes/CpgSeq/2009-1-22: Difference between revisions
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===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 === | ===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 === | ||
reaction system | reaction system x14 x2 | ||
H2O 42.6ul | H2O 42.6ul 596.4ul | ||
2x Master mix 50ul | 2x Master mix 50ul 700ul | ||
dUTP(1mM) 2ul | dUTP(1mM) 2ul 28ul | ||
AmpF6.3(10uM) 2ul | AmpF6.3(10uM) 2ul 28ul | ||
AmpR6.3(10uM) 2ul | AmpR6.3(10uM) 2ul 28ul | ||
50x SYBG I 0.4ul | 50x SYBG I 0.4ul 5.6ul | ||
template(4ng/ul) 0.5ul 2 uleach | template(4ng/ul) 0.5ul 2 uleach | ||
Total 100ul | Total 100ul 1400ul | ||
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C | 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C |
Revision as of 22:31, 28 January 2009
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 reaction
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 0.2 (10mM)0.02ul x100 58ul 10ul 10ul 20ul 2ul
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(01/26/2009)
x12 Template 10ul 2X iProof Mastermix 50ul 600ul AmpF6.2SoL (10uM) 4ul 48ul AmpR6.2SoL (10uM) 4ul 48ul 50X SYBG I 0.8ul 9.6ul H2O 31.2ul 374.4ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
File:20090126 No 21 22.jpg20090126_No_21_22
File:20090126 No 23 24 25 26.jpg20090126_No_23_24_25_26
quantification of gel purified amplicon
no_21 PGP1_iPS1: cpg30k 1ng/ul x 10ul; V2.1 1ng/ul x 10ul; No_22 PGP1_L: cpg30k 3ng/ul x 10ul;
No_23 PGP3_L: cpg30k 3ng/ul x 10ul; V2.1 3 ng/ul x 10ul; V2.2 1ng/ul x 10ul; V2.3 3ng/ul x 10ul;
No_24 PGP9_L: cpg30k 3ng/ul x 10ul; V2.1 3 ng/ul x 10ul; V2.3 3ng/ul x 10ul;
No_25 PGP1F.2: cpg30k 2ng/ul;
No_26 PGP9F.2: cpg30k 2ng/ul;
no_21 PGP1_iPS1: cpg30k 1ng/ul 2ul + V2.1 1ng/ul x 2ul; No_22 PGP1_L: cpg30k 3ng/ul x 2ul; No_23 PGP3_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.2 1ng/ulx 2ul + V2.3 3ng/ul x 1ul; No_24 PGP9_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.3 3ng/ul x 1ul; No_25 PGP1F.2: cpg30k 2ng/ul x 2ul; No_26 PGP9F.2: cpg30k 2ng/ul x 2ul;
sequencing library construction
2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40
reaction system x14 x2 H2O 42.6ul 596.4ul 2x Master mix 50ul 700ul dUTP(1mM) 2ul 28ul AmpF6.3(10uM) 2ul 28ul AmpR6.3(10uM) 2ul 28ul 50x SYBG I 0.4ul 5.6ul template(4ng/ul) 0.5ul 2 uleach Total 100ul 1400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Bioneer column purification. Yield:
Digestion with MmeI
No_4_5_15 x3 Total 30ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 12 1mM SAM(fresh) 4ul 12 2U/ul Mme I 8ul 24 ddH2O 4ul 12 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB.
step3. USER digestion
DNA 10ul USER 3ul total 13ul
37C 2h.
step4. S1 nuclease digestion
No4_5_15 x3 10 x S1 nuclease buffer: 2ul 6ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 3ul ddH2O 4ul 12ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
step5. end repair
No4_5_15 positive control x4 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 10 dATP 2.5ul 2.5ul 10 10xendrepair buffer 2.5ul 2.5ul 10 enzyme 0.5ul 0.5ul 2 extra ATP(10mM) 2.5ul 2.5ul 10
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
step6. adapter ligation
No4_5_15 positive control negative control x5 DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 2.5 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 2.5 2xQuickLiage buffer 15ul 15ul 15ul 75 QuickLigase enzyme(NEB) 1ul 1ul 1ul 5 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 12.5
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.
step7. Nick-translation
set up the ligation system: x5 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 10ul 10mM dNTP 0.4ul 2ul 1mg/ml BSA 2ul 10ul Bst polymerase(8U/ul) 1ul 5ul
65C for 25 minutes -> keep on ice.
step8. PCR of sequencig library
x5 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 10ul Solexa_PCR_lo(10uM) 2ul 10ul 2xiProof master mix 50ul 250ul 50x SYBG 0.8ul 4ul ddH2O 25.2ul 126ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.