Jie:LabNotes/CpgSeq/2008-7-31

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Revision as of 16:17, 31 July 2008 by >Jie deng
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sequencing library construction

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

 reaction system                                                x4       
 H2O                                                43.2ul     172.8ul  
 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(18.4ng/ul)                                 0.2ul      0.8ul
 Total                                               100ul      400ul
 94C 3min -> 11cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Bioneer column purification. Yield:30.4ng/ul x34ul

step2. Digestion with MmeI

                        dUTP 250uM,167.3ng/ul       dUTP 100uM, 159.8ng/ul (06/17/2008)
 Total                               20ul    
 DNA                                  6ul            6ul
 10X NEBuffer 4                       2ul            4ul
 1mM SAM(fresh)                       2ul            4ul
 2U/ul Mme I                          8ul            8ul
 ddH2O                                2ul            2ul  
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
 MinElute column purify. Elute in 11ul EB.

step3. USER digestion

 DNA                    10ul         
 USER                    3ul        
 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

 Total                  25ul                        
 DNA                    17ul                         
 dNTP                  2.5ul           
 dATP                  2.5ul                 
 10xendrepair buffer   2.5ul             
 enzyme                0.5ul          
 
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation

 total                       30ul        control            
 DNA                         13ul       ddH2O 13ul            
 100uM Solexa_1 adaptor     0.5ul         0.5ul 
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 
 2xQuickLiage buffer         15ul          15ul    
 QuickLigase enzyme(NEB)      1ul           1ul      
  
 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