Jie:LabNotes/CpgSeq/2008-8-15
2rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit[edit]
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 | |
1 | BJ | 178ng/ul x 2 tubes | 11.2ul | 8.8ul | 130ul | 43.3ng/ul x 20ul | 2.18/2.97 |
2 | BJ_iPS_11 | 140ng/ul x 2 tubes | 14.3ul | 5.7ul | 130ul | 65.6ng/ul x 20ul | 2.19/2.91 |
6 | IMB90 | 50ng/ul | 20ul | 0ul | 130ul | 22.7ng/ul x 10ul | 2.05/2.78 |
9 | Hybrid2 | 200ng/ul | 10ul | 10ul | 130ul | 174.5ng/ul x 10ul | 2.3/3.0 |
12 | Hues8 | 178.5ng/ul | 13ul | 7ul | 130ul | 8ng/ul x 10ul | 1.37/1.01 |
13 | Hues42 | 20ng/ul x 2 tube | 20ul | 0ul | 130ul | 14ng/ul x 20ul |
- 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[edit]
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 | |
1 | BJ | 43.3ng/ul x 20ul | 1ul | 2+1+1+5ul | 3+1.5+4.5 | 5+1.5+2.5 | 4+1.5+3.5 |
2 | BJ_iPS_11 | 65.6ng/ul x 20ul | 1ul | 2+1+1+5ul | 3+1.5+4.5 | 5+1.5+2.5 | 4+1.5+3.5 |
3 | BJ-iPS_12 | 43.3ng/ul x 20ul | 1ul | 2+1+1+5ul | 3+1.5+4.5 | 5+1.5+2.5 | 4+1.5+3.5 |
6 | IMB90 | 22.9ng/ul x 30ul | 1ul | 4+1+1+3ul | |||
9 | Hybrid_2 | 174.5ng/ul x 10ul + 65.6 x 10ul | 1ul | 2+1+1+5ul | 3+1.5+4.5 | 5+1.5+2.5 | 4+1.5+3.5 |
positive control | 200ng/ul | 1ul | 1+1+1+6ul | 1+1.5+6.5 | 2+1.5+5.5 | 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(08/16/2008)[edit]
x8 Template 10ul 2X iProof Mastermix 50ul 400ul AmpF6.2SoL (10uM) 4ul 32ul AmpR6.2SoL (10uM) 4ul 32ul 50X SYBG I 0.8ul 6.4ul H2O 31.2ul 249.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
File:20080817 PCR No1 No2.jpg20080817_PCR_No1_No2
File:20080817 PCR No3 No9.jpg20080817_PCR_No3_No9
since the cpg30kV2.2 were not amplified, I redo the bisulfite conversion and capture of cpg30kV2.2.
3rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit(2008-08-29)[edit]
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | |
1 | BJ | 178ng/ul | 11.2ul | 8.8ul | 130ul | 48.1ng/ul x 10ul |
2 | BJ_iPS_11 | 140ng/ul | 14.3ul | 5.7ul | 130ul | 74.8ng/ul x 10ul |
3 | BJ_iPS_12 | 40ng/ul | 20ul | 0ul | 130ul | 41.4ng/ul x 10ul |
9 | Hybrid2 | 200ng/ul | 10ul | 10ul | 130ul | 122.1ng/ul x 10ul |
Jurkat | 154ng/ul x 2 tube | 10ul | 10ul | 130ul | 201.4ng/ul x 20ul |
- 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[edit]
sample | sample concentration | 10xLigase buffer | template+v2.2(21ng/ul) vol+H2O | |
1 | BJ | 48.1ng/ul x 10ul | 1.5ul | 10+1.5+2.5 |
2 | BJ_iPS_11 | 74.8ng/ul x 10ul | 1.5ul | 10+1.5+2.5 |
3 | BJ-iPS_12 | 41.4ng/ul x 10ul | 1.5ul | 10+1.5+2.5 |
9 | Hybrid_2 | 122.1ng/ul x 10ul | 1.5ul | 10+1.5+2.5 |
positive control_Jurkat | 201.4ng/ul | 1.5ul | 2+1.5+5.5 | |
negative control | 0 | 1.5ul | 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 0.2ul (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(09/01/2008)[edit]
x6 Template 10ul 2X iProof Mastermix 50ul 300ul AmpF6.2SoL (10uM) 4ul 24ul AmpR6.2SoL (10uM) 4ul 24ul 50X SYBG I 0.8ul 4.8ul H2O 31.2ul 187.2ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
File:20080901 cpg30kV2.2 captured PCR No 1 2 3 9 Jurkat.jpg20080902_cpg30kV2.2_captured PCR_No1_2_3_9_Jurkat
File:20080901 Quantification of No1 2.jpg20080901_quantification of No_1_2
File:20080901 quantification of No 3 9.jpg20080901_quantificatio of No3_9
yield: No_1: cpg30k + suppressor: 6ng/ul x 15ul V2.1: 6ng/ul x 15ul V2.2: 4ng/ul x 15ul V2.3: 6ng/ul x 15ul V2.2_09/01/08: 3ng/ul x 15ul mix 5ul cpg30k + suppressor + 5ul V2.1 + 10ul V2.2_09/01/08 + 5ul V2.3 = 4.8ng/ul x 25ul
No_2: cpg30k + suppressor: 6ng/ul x 15ul V2.1: 6ng/ul x 15ul V2.2: 3ng/ul x 15ul V2.3: 5ng/ul x 15ul V2.2_09/01/08: 4ng/ul x 15ul mix 5ul cpg30k + suppressor + 5ul V2.1 + 7.5ul V2.2_09/01/08 + 6ul V2.3 = 5ng/ul x 23.5ul
No_3:
cpg30k + suppressor: 4.5ng/ul x 15ul V2.1: 4.5ng/ul x 15ul V2.2: 2.5ng/ul x 15ul V2.3: 2.5ng/ul x 15ul V2.2_09/01/08: 2ng/ul x 15ul mix 5ul cpg30k + suppressor + 5ul V2.1 + 4ul V2.2 + 9ulV2.3 + 5ul V2.2_09/01/08 = 3.2ng/ul x 28ul
No_9:
cpg30k + suppressor: 5ng/ul x 15ul V2.1: 3ng/ul x 15ul V2.2: 1.5ng/ul x 15ul V2.3: 2ng/ul x 15ul V2.2_09/01/08: 3ng/ul x 15ul mix 4ul cpg30k + suppressor + 7ul V2.1 + 10ul V2.3 + 7ul V2.2_09/01/08 = 2.8ng/ul x 28ul
sequencing library construction[edit]
2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40[edit]
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 Total 100ul 400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Bioneer column purification. Yield: No_1: 33.2ng/ul x 30ul No_2: 39.4ng/ul x 30ul No_3: 56.7ng/ul x 30ul No_9: 49.4ng/ul x 30ul
Digestion with MmeI[edit]
No_1_2_3_9 x4 Total 30ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 16 1mM SAM(fresh) 4ul 16 2U/ul Mme I 8ul 32 ddH2O 4ul 16 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB.
File:20080901 dUTP PCR and MmeI.jpg20080901_dUTP_PCR and MmeI
step3. USER digestion[edit]
DNA 10ul USER 3ul total 13ul
37C 12h.
step4. S1 nuclease digestion[edit]
No1_2_3_9 x4 10 x S1 nuclease buffer: 2ul 8ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 4ul ddH2O 4ul 16ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
step5. end repair[edit]
No1_2_3_9 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.
step6. adapter ligation[edit]
No1_2_3_9 positive control negative control x6 DNA 13ul 13ul ddH2O 13ul 48 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 3 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 3 2xQuickLiage buffer 15ul 15ul 15ul 90 QuickLigase enzyme(NEB) 1ul 1ul 1ul 6 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 15
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.
File:20080902 gul size selectin after adaptor ligation.jpg20080902_gel size selection after adaptor ligation
File:20080902 positive and negative control of adaptor ligation.jpg20080902_gel of control and genative control
step7. Nick-translation[edit]
set up the ligation system: x6 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 12ul 10mM dNTP 0.4ul 2.4ul 1mg/ml BSA 2ul 12ul Bst polymerase(8U/ul) 1ul 6ul
65C for 25 minutes -> keep on ice.
step8. PCR of sequencig library[edit]
x6 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 12ul Solexa_PCR_lo(10uM) 2ul 12ul 2xiProof master mix 50ul 300ul 50x SYBG 0.8ul 4.8ul ddH2O 25.2ul 151.2ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
File:20080902 sequencing library of No1 2 3 9.jpg20080902_sequencing library of No1_2_3_9
Qiaquick column purification. Yield:
No_1: BJ, 17.4ng/ul (117nM) x 30ul No_2: BJ_iPS_11, 18.6ng/ul (125nM) x 30ul No_3: BJ_iPS_12, 32.7ng/ul (220nM) x 30ul No_9: Hybrid_2, 28.3ng/ul (190nM) x 30ul
Sent 15ul of each sequencing library to Yuan Gao on 08-09-02.
Blunt clone and transformation of captured PCR products and random sequencing library of No1_BJ (2008-09-04)[edit]
refer to LabNotes on [1]