Jie:LabNotes/CpgSeq/2008-9-5
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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
quantification of PCR products
yield: No_4:
cpg30k + suppressor: 5ng/ul x 15ul V2.1: 5ng/ul x 15ul V2.2: 4ng/ul x 15ul V2.3: 4.5ng/ul x 15ul mix 3ul cpg30K + 3ul V2.1 + 4ul V2.2 + 3ul V2.3 = 5ng/ul x 13ul
No_5: cpg30k + suppressor: 4ng/ul x 15ul V2.1: 5ng/ul x 15ul V2.2: 3ng/ul x 15ul V2.3: 3ng/ul x 15ul mix 3ul cpg30k + 2.5ul V2.1 + 4ul V2.2 + 4ul V2.3 = 4ng/ul x 14.5ul
No_15: cpg30k + suppressor: 6ng/ul x 15ul V2.1: 6ng/ul x 15ul V2.2: 4.5ng/ul x 15ul V2.3: 4ng/ul x 15ul mix 3ul cpg30k + 3ul V2.1 + 4ul V2.2 + 4.5ul V2.3 = 4.5ng/ul x 14.5ul
No_6: (2008-09-14) cpg30k + suppressor: 9ng/ul V2.1: 9ng/ul mix 4ul cpg30k + 3ul V2.1 = 9ng/ul x 7ul
sequencing library construction
2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40
reaction system x4 H2O 42.6ul 170.4ul 2x Master mix 50ul 200ul dUTP(1mM) 2ul 8ul AmpF6.3(10uM) 2ul 8ul AmpR6.3(10uM) 2ul 8ul 50x SYBG I 0.4ul 1.6ul template(4ng/ul) 0.5ul 2ul each Total 100ul 400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Bioneer column purification. Yield: No_4: 52.8ng/ul x 30ul No_5: 33.1ng/ul x 30ul No_15: 42.2ng/ul x 30ul No_6: (2008-09-14)
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.
File:200809010 dUTP-PCR MmeI No 4 5 15.jpg200809010_dUTP-PCR_MmeI_No_4_5_15
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.
File:20080912 sequencing library No 4 5 15.jpg20080912_sequencing library_No_4_5_15
Qiaquick column purification. Yield:
No_4: hFib2, 27.1ng/ul (164nM) x 30ul No_5: hFib2_iPS4, 22.2ng/ul (134nM) x 30ul No_15: HUES_63, 30.8ng/ul (186nM) x 30ul