Jie:LabNotes/CpgSeq/2008-7-31: Difference between revisions

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Line 56: Line 56:
   10xendrepair buffer  2.5ul          2.5ul   
   10xendrepair buffer  2.5ul          2.5ul   
   enzyme                0.5ul          0.5ul
   enzyme                0.5ul          0.5ul
   extra ATP             2.5ul          2.5ul  
   extra ATP(10mM)      2.5ul          2.5ul  


   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
Line 62: Line 62:
==step6. adapter ligation==
==step6. adapter ligation==
                            
                            
   total                      30ul       control          
   total                      30ul   positive control     negative control     
   DNA                        13ul       ddH2O 13ul             
   DNA                        13ul         13ul            ddH2O 13ul             
   100uM Solexa_1 adaptor    0.5ul        0.5ul  
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul
   100uM Solexa_2 adaptor    0.5ul        0.5ul                 
   100uM Solexa_2 adaptor    0.5ul        0.5ul                0.5ul
   2xQuickLiage buffer        15ul          15ul  
   2xQuickLiage buffer        15ul          15ul                 15ul
   QuickLigase enzyme(NEB)      1ul          1ul    
   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 EB.
   Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.


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

Revision as of 22:38, 1 August 2008

sequencing library construction

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

 reaction system                                                x4         x3 
 H2O                                                43.2ul     172.8ul  518.4
 2x Master mix                                        50ul      200ul     600
 dUTP(1mM)                                             2ul        8ul      24
 AmpF6.3(10uM)                                         2ul        8ul      24
 AmpR6.3(10uM)                                         2ul        8ul      24
 50x SYBG I                                          0.4ul      1.6ul     4.8
 template(18.4ng/ul)                                 0.1ul      0.4ul     1.2
 Total                                               100ul      400ul     2400
 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.

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.

step5. end repair

                                   positive control
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_sapcer_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 EB.
 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)           1ul            3ul  
 Solexa_PCR_lo(10uM)           1ul            3ul
 2xiProof master mix          25ul           75ul  
 50x SYBG                    0.4ul          1.2ul
 ddH2O                      12.6ul         37.8ul
 98C 30sec -> 13 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.


Purify with Qiaquick column. Elute in 30ul EB. dUTP250uM: 12.8ng/ul dUTP100uM: 22.5ng/ul