Alice:LabNotes/2009-1-13

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Revision as of 22:39, 14 January 2009 by >Jie deng (→‎step4. S1 nuclease digestion)
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Construct Solexa sequencing library

2nd PCR amplification

 AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 
 system setup:                                                   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(19ng/ul)                                 0.1ul        0.4ul     
 Total                                               100ul      400ul     
 94C 3min -> 9 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Qiaquick column purification.
 30ul * 47ng/ul

step2. Digestion with MmeI

 (dUTP 250uM,167.3ng/ul of product will use the system below)      
 Total                               20ul      
 DNA                                  6ul            
 10X NEBuffer 4                       2ul           
 1mM SAM(fresh)                       2ul          
 2U/ul Mme I                          8ul          
 ddH2O                                2ul          
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
 however, since the concentration I obtained from previous is 47ng/ul, therefore the following is added:
 DNA              14ul
 SAM(1mM)          4ul
 NEBuffer 4        2ul
 37C 2hr
 
MinElute column purify. Elute in 22ul EB.

step3. USER digestion

 DNA                    10ul         
 USER                    3ul     
 total                  13ul           
 37C 4hr

step4. S1 nuclease digestion

 10 x S1 nuclease buffer:   2ul     
 DNA after USER digestion: 13ul    
 S1 nuclease (10U/ul):      1ul     
 ddH2O                      4ul     
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

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.


 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.

Purify with Qiaquick column. Elute in 30ul EB.