Jie:LabNotes/CpgSeq/2009-1-13: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Jie deng
No edit summary
>Jie deng
No edit summary
Line 2: Line 2:


   No1: BJ (4.8ng/ul, 09/01/08);
   No1: BJ (4.8ng/ul, 09/01/08);
   NO8: Hybrid_1 (4ng/ul, 09/01/08);
   NO8: Hybrid_1 (4ng/ul, 08/04/08);
   No9: Hybrid_2 (2.8ng/ul, 09/01/08);
   No9: Hybrid_2 (2.8ng/ul, 09/01/08);
   No17: PGPF1 (11/04/08);
   No17: PGPF1 (7ng/ul, 10/10/08);
   No18: Hues12 (11/04/08).
   No18: Hues12 (6ng/ul, 11/04/08).


==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 ==

Revision as of 20:02, 13 January 2009

sequencing library construction (2009-01-13) of BJ, Hybrid_1, Hybrid_2, PGP1F, Hues12

 No1: BJ (4.8ng/ul, 09/01/08);
 NO8: Hybrid_1 (4ng/ul, 08/04/08);
 No9: Hybrid_2 (2.8ng/ul, 09/01/08);
 No17: PGPF1 (7ng/ul, 10/10/08);
 No18: Hues12 (6ng/ul, 11/04/08).

2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40

 reaction system                                                x4         x5 
 H2O                                                43.2ul     172.8ul    864
 2x Master mix                                        50ul      200ul    1000
 dUTP(1mM)                                             2ul        8ul      40
 AmpF6.3(10uM)                                         2ul        8ul      40
 AmpR6.3(10uM)                                         2ul        8ul      40
 50x SYBG I                                          0.4ul      1.6ul       8
 template(18.4ng/ul)                                 0.1ul      0.4ul       2
 Total                                               100ul      400ul     2000
 94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Bioneer column purification. Yield:167ng/ul x60ul

step2. Digestion with MmeI

                        dUTP 250uM,167.3ng/ul      x2 
 Total                               20ul      
 DNA                                  6ul          12  
 10X NEBuffer 4                       2ul           4 
 1mM SAM(fresh)                       2ul           4
 2U/ul Mme I                          8ul          16 
 ddH2O                                2ul           4 
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
 MinElute column purify. Elute in 22ul EB.

File:20080801 after MmeI.jpg20080801_after MmeI

step3. USER digestion

                                 x2
 DNA                    10ul      20ul   
 USER                    3ul       6ul  
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion

                                   x2
 10 x S1 nuclease buffer:   2ul     4ul
 DNA after USER digestion: 13ul    26ul
 S1 nuclease (10U/ul):      1ul     2ul
 ddH2O                      4ul     8ul
 37C 10mins.
 Minelute cloumn purify. Elute in 18ul H2O.

File:20080801 after USER and S1.jpg20080801_after USER and S1

step5. end repair

                                   positive control
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul
 dATP                  2.5ul           2.5ul      
 10xendrepair buffer   2.5ul           2.5ul  
 enzyme                0.5ul           0.5ul
 extra ATP(10mM)       2.5ul           2.5ul 
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation

 total                       30ul    positive control     negative control       
 DNA                         13ul          13ul            ddH2O 13ul            
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 0.5ul
 2xQuickLiage buffer         15ul          15ul                  15ul
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul
 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O.

File:20080801 after endrepair and ligation.jpg

 PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.

step7. Nick-translation

 set up the ligation system:
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul          
 10mM dNTP                  0.4ul           
 1mg/ml BSA                   2ul            
 Bst polymerase(8U/ul)        1ul             
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library

                                               x3
 Nick-translated DNA          10ul             
 Solexa_PCR_up(10uM)           2ul            6ul  
 Solexa_PCR_lo(10uM)           2ul            6ul
 2xiProof master mix          50ul          150ul  
 50x SYBG                    0.8ul          2.4ul
 ddH2O                      35.2ul         106.6ul
 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20080803 sequencing library.jpg20080803_sequencing library

Purify with Qiaquick column. Elute in 30ul EB. yield: 22.9ng/ul(173nM)