Jie:LabNotes/CpgSeq/2008-9-12: Difference between revisions

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(New page: ==sequencing library construction for paralleling sequencing == Use the captured PCR mix of No_1(BJ) and No_9(Hybrid_2) as the templates. ==2nd PCR amplification with AmpF6.3/AmpR6.3 an...)
 
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==sequencing library construction for paralleling sequencing ==
==sequencing library construction for paralleling sequencing ==


Use the captured PCR mix of No_1(BJ) and No_9(Hybrid_2) as the templates.  
Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-8-15]


==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 16:28, 12 September 2008

sequencing library construction for paralleling sequencing

Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [1]

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

 reaction system                                      x4     
 H2O                                                42.6ul     170.4ul    
 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(4ng/ul)                                    0.5ul        2ul     
 Total                                               100ul      400ul      
 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 Bioneer column purification. Yield:
 No_1: 33.2ng/ul x 30ul 
 No_9: 49.4ng/ul x 30ul

Digestion with MmeI

                                     No_1_2_3_9    x4 
 Total                                30ul                  
 dUTP_PCR                             20ul         
 10X NEBuffer 4                       4ul          16           
 1mM SAM(fresh)                       4ul          16             
 2U/ul Mme I                          8ul          32               
 ddH2O                                4ul          16
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h. MinEulte column purification. Elute in 12ul EB.

step3. USER digestion

 DNA                    10ul         
 USER                    3ul        
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion

                            No1_2_3_9      x4 
 10 x S1 nuclease buffer:   2ul            8ul
 DNA after USER digestion: 13ul            13ul
 S1 nuclease (10U/ul):      1ul            4ul
 ddH2O                      4ul            16ul
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

step5. end repair

                      No1_2_3_9         positive control               x5
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul                           12.5 
 dATP                  2.5ul           2.5ul                           12.5
 10xendrepair buffer   2.5ul           2.5ul                           12.5 
 enzyme                0.5ul           0.5ul                            2.5
 extra ATP(10mM)       2.5ul           2.5ul                           12.5
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation

                          No1_2_3_9    positive control     negative control       x6
 DNA                         13ul          13ul            ddH2O 13ul              48  
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul               3
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 0.5ul               3
 2xQuickLiage buffer         15ul          15ul                  15ul              90
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul               6
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul              15  
 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O.
 TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.

step7. Nick-translation

 set up the ligation system:
                                          x6              
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul         12ul 
 10mM dNTP                  0.4ul        2.4ul    
 1mg/ml BSA                   2ul         12ul   
 Bst polymerase(8U/ul)        1ul          6ul    
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library

                                               x6
 Nick-translated DNA          20ul             
 Solexa_PCR_up(10uM)           2ul            12ul  
 Solexa_PCR_lo(10uM)           2ul            12ul
 2xiProof master mix          50ul           300ul  
 50x SYBG                    0.8ul           4.8ul
 ddH2O                      25.2ul         151.2ul
 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.