Jie:LabNotes/CpgSeq/2008-9-12: Difference between revisions
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==sequencing library construction for | ==parallel sequencing library construction for No_1(BJ) and No_9(Hybrid_2)== | ||
Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-8-15] | Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-8-15] | ||
Line 12: | Line 12: | ||
AmpR6.3(10uM) 2ul 8ul | AmpR6.3(10uM) 2ul 8ul | ||
50x SYBG I 0.4ul 1.6ul | 50x SYBG I 0.4ul 1.6ul | ||
template(4ng/ul) 0.5ul | template(4ng/ul) 0.1ul 0.5ul | ||
Total 100ul 400ul | Total 100ul 400ul | ||
94C 3min -> | 94C 3min -> 11 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C | ||
Bioneer column purification. Yield: | Bioneer column purification. Yield: | ||
No_1: | No_1: 30.5ng/ul x 30ul | ||
No_9: | No_9: 44.7ng/ul x 30ul | ||
==Digestion with MmeI== | ==Digestion with MmeI== | ||
No_1_9 x2 | |||
Total 30ul | Total 30ul | ||
dUTP_PCR 20ul | dUTP_PCR 20ul | ||
10X NEBuffer 4 4ul | 10X NEBuffer 4 4ul 8 | ||
1mM SAM(fresh) 4ul | 1mM SAM(fresh) 4ul 8 | ||
2U/ul Mme I 8ul | 2U/ul Mme I 8ul 16 | ||
ddH2O 4ul | ddH2O 4ul 8 | ||
1mM SAM: 32mM SAM 1ul + 31ul ddH2O. | 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. | ||
37C 2h. MinEulte column purification. Elute in 12ul EB. | 37C 2h. MinEulte column purification. Elute in 12ul EB. | ||
[[Image:20080912_dUTP_PCR and MmeI of No_1_9.jpg]]20080912_dUTP_PCR and MmeI of No_1_9 | |||
==step3. USER digestion== | ==step3. USER digestion== | ||
Line 42: | Line 44: | ||
==step4. S1 nuclease digestion== | ==step4. S1 nuclease digestion== | ||
No1_9 x2 | |||
10 x S1 nuclease buffer: 2ul | 10 x S1 nuclease buffer: 2ul 4ul | ||
DNA after USER digestion: 13ul 13ul | DNA after USER digestion: 13ul 13ul | ||
S1 nuclease (10U/ul): 1ul | S1 nuclease (10U/ul): 1ul 2ul | ||
ddH2O 4ul | ddH2O 4ul 8ul | ||
37C 10mins. | 37C 10mins. | ||
Line 52: | Line 54: | ||
==step5. end repair== | ==step5. end repair== | ||
No1_9 positive control x3 | |||
Total 25ul H2O 13ul | Total 25ul H2O 13ul | ||
DNA 15ul 2ul(20080801_spacer_107bp) | DNA 15ul 2ul(20080801_spacer_107bp) | ||
dNTP 2.5ul 2.5ul | dNTP 2.5ul 2.5ul 7.5 | ||
dATP 2.5ul 2.5ul | dATP 2.5ul 2.5ul 7.5 | ||
10xendrepair buffer 2.5ul 2.5ul | 10xendrepair buffer 2.5ul 2.5ul 7.5 | ||
enzyme 0.5ul 0.5ul | enzyme 0.5ul 0.5ul 1.5 | ||
extra ATP(10mM) 2.5ul 2.5ul | extra ATP(10mM) 2.5ul 2.5ul 7.5 | ||
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul | Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 28ul H2O. Take 14ul to do the adapter ligation. | ||
==step6. adapter ligation== | ==step6. adapter ligation== | ||
adaptor constraction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 95C 5mins -> 65C 30mins -> 4C. | |||
No1_9 positive control negative control x4 | |||
DNA 13ul 13ul ddH2O 13ul | DNA 13ul 13ul ddH2O 13ul | ||
100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul | 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 2 | ||
100uM | 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 2 | ||
2xQuickLiage buffer 15ul 15ul 15ul | 2xQuickLiage buffer 15ul 15ul 15ul 60 | ||
QuickLigase enzyme(NEB) 1ul 1ul 1ul | QuickLigase enzyme(NEB) 1ul 1ul 1ul 4 | ||
extra ATP(10mM) 2.5ul 2.5ul 2.5ul | extra ATP(10mM) 2.5ul 2.5ul 2.5ul 10 | ||
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O. | 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. | TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O. | ||
[[Image:20080913_gel cut after adaptor ligation.jpg]]20080913_gel cut after adaptor ligation | |||
==step7. Nick-translation== | ==step7. Nick-translation== | ||
set up the ligation system: | set up the ligation system: | ||
x4 | |||
Total 20ul | Total 20ul | ||
DNA 15ul | DNA 15ul | ||
10x ThermoPol buffer 2ul | 10x ThermoPol buffer 2ul 8ul | ||
10mM dNTP 0.4ul | 10mM dNTP 0.4ul 1.6ul | ||
1mg/ml BSA 2ul | 1mg/ml BSA 2ul 8ul | ||
Bst polymerase(8U/ul) 1ul | Bst polymerase(8U/ul) 1ul 4ul | ||
65C for 25 minutes -> keep on ice. | 65C for 25 minutes -> keep on ice. | ||
==step8. PCR of sequencig library== | ==step8. PCR of sequencig library== | ||
x4 | |||
Nick-translated DNA 20ul | Nick-translated DNA 20ul | ||
Solexa_PCR_up(10uM) 2ul | Solexa_PCR_up(10uM) 2ul 8ul | ||
Solexa_PCR_lo(10uM) 2ul | Solexa_PCR_lo(10uM) 2ul 8ul | ||
2xiProof master mix 50ul | 2xiProof master mix 50ul 200ul | ||
50x SYBG 0.8ul | 50x SYBG 0.8ul 3.2ul | ||
ddH2O 25.2ul | ddH2O 25.2ul 100.8ul | ||
98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. | 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. | ||
[[Image:20080913_parellel sequencing library of No_1_9.jpg]]20080913_parellel sequencing library of No_1_9 | |||
Qiaquick column purification. Yields: | |||
No_1(BJ): 17.2ng/ul (104nM) x 30ul | |||
No_9(Hybrid_2): 18.6ng/ul (112nM) x 30ul | |||
Send 15ul of each sample to Bing Ren on 09/15/08. | |||
==Blunt cloning of sequencing library and sequencing (2008-10-16)== | |||
refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-10-16] |
Latest revision as of 16:54, 16 October 2008
parallel sequencing library construction for No_1(BJ) and No_9(Hybrid_2)[edit]
Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [1]
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) 0.1ul 0.5ul Total 100ul 400ul
94C 3min -> 11 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Bioneer column purification. Yield: No_1: 30.5ng/ul x 30ul No_9: 44.7ng/ul x 30ul
Digestion with MmeI[edit]
No_1_9 x2 Total 30ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 8 1mM SAM(fresh) 4ul 8 2U/ul Mme I 8ul 16 ddH2O 4ul 8 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB.
File:20080912 dUTP PCR and MmeI of No 1 9.jpg20080912_dUTP_PCR and MmeI of No_1_9
step3. USER digestion[edit]
DNA 10ul USER 3ul total 13ul
37C 12h.
step4. S1 nuclease digestion[edit]
No1_9 x2 10 x S1 nuclease buffer: 2ul 4ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 2ul ddH2O 4ul 8ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
step5. end repair[edit]
No1_9 positive control x3 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 7.5 dATP 2.5ul 2.5ul 7.5 10xendrepair buffer 2.5ul 2.5ul 7.5 enzyme 0.5ul 0.5ul 1.5 extra ATP(10mM) 2.5ul 2.5ul 7.5
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 28ul H2O. Take 14ul to do the adapter ligation.
step6. adapter ligation[edit]
adaptor constraction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 95C 5mins -> 65C 30mins -> 4C. No1_9 positive control negative control x4 DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 2 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 2 2xQuickLiage buffer 15ul 15ul 15ul 60 QuickLigase enzyme(NEB) 1ul 1ul 1ul 4 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 10
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:20080913 gel cut after adaptor ligation.jpg20080913_gel cut after adaptor ligation
step7. Nick-translation[edit]
set up the ligation system: x4 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 8ul 10mM dNTP 0.4ul 1.6ul 1mg/ml BSA 2ul 8ul Bst polymerase(8U/ul) 1ul 4ul
65C for 25 minutes -> keep on ice.
step8. PCR of sequencig library[edit]
x4 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 8ul Solexa_PCR_lo(10uM) 2ul 8ul 2xiProof master mix 50ul 200ul 50x SYBG 0.8ul 3.2ul ddH2O 25.2ul 100.8ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
File:20080913 parellel sequencing library of No 1 9.jpg20080913_parellel sequencing library of No_1_9
Qiaquick column purification. Yields:
No_1(BJ): 17.2ng/ul (104nM) x 30ul No_9(Hybrid_2): 18.6ng/ul (112nM) x 30ul
Send 15ul of each sample to Bing Ren on 09/15/08.
Blunt cloning of sequencing library and sequencing (2008-10-16)[edit]
refer to LabNotes on [2]