Alice:LabNotes/2009-1-13: Difference between revisions

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>Jie deng
>Jie deng
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==2nd PCR amplification==
==2nd PCR amplification==
   AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40  
   AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40  
   system setup:                                             x4         x3
   system setup:                                                   x4        
   H2O                                                43.2ul    172.8ul  518.4
   H2O                                                43.2ul    172.8ul   
   2x Master mix                                        50ul      200ul    600
   2x Master mix                                        50ul      200ul     
   dUTP(1mM)                                            2ul        8ul      24
   dUTP(1mM)                                            2ul        8ul       
   AmpF6.3(10uM)                                        2ul        8ul      24
   AmpF6.3(10uM)                                        2ul        8ul       
   AmpR6.3(10uM)                                        2ul        8ul     24
   AmpR6.3(10uM)                                        2ul        8ul    
   50x SYBG I                                          0.4ul      1.6ul     4.8
   50x SYBG I                                          0.4ul      1.6ul      
   template(18.4ng/ul)                                0.1ul     0.4ul    1.2
   template(19ng/ul)                                0.1ul       0.4ul     
   Total                                              100ul      400ul    2400
   Total                                              100ul      400ul     


   94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
   94C 3min -> 9 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
    
    
   Bioneer column purification. Yield:167ng/ul x60ul
   Bioneer column purification.


==step2. Digestion with MmeI==
==step2. Digestion with MmeI==

Revision as of 21:19, 13 January 2009

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
 
 Bioneer column purification.

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_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.