Jie:LabNotes/CpgSeq/2008-9-12

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parallel sequencing library construction for No_1(BJ) and No_9(Hybrid_2)[edit]

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[edit]

 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)                                    0.1ul      0.5ul     
 Total                                               100ul      400ul      
 94C 3min -> 11 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 Bioneer column purification. Yield:
 No_1: 30.5ng/ul x 30ul 
 No_9: 44.7ng/ul x 30ul

Digestion with MmeI[edit]

                                     No_1_9        x2 
 Total                                30ul                  
 dUTP_PCR                             20ul         
 10X NEBuffer 4                       4ul          8           
 1mM SAM(fresh)                       4ul          8             
 2U/ul Mme I                          8ul          16               
 ddH2O                                4ul          8
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h. MinEulte column purification. Elute in 12ul EB.

File:20080912 dUTP PCR and MmeI of No 1 9.jpg20080912_dUTP_PCR and MmeI of No_1_9

step3. USER digestion[edit]

 DNA                    10ul         
 USER                    3ul        
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion[edit]

                            No1_9          x2 
 10 x S1 nuclease buffer:   2ul            4ul
 DNA after USER digestion: 13ul            13ul
 S1 nuclease (10U/ul):      1ul            2ul
 ddH2O                      4ul            8ul
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

step5. end repair[edit]

                      No1_9         positive control                    x3
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul                           7.5 
 dATP                  2.5ul           2.5ul                           7.5
 10xendrepair buffer   2.5ul           2.5ul                           7.5 
 enzyme                0.5ul           0.5ul                           1.5
 extra ATP(10mM)       2.5ul           2.5ul                           7.5
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 28ul H2O. Take 14ul to do the adapter ligation.

step6. adapter ligation[edit]

 adaptor constraction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 95C 5mins -> 65C 30mins -> 4C.
                         
                          No1_9     positive control     negative control          x4
 DNA                         13ul          13ul            ddH2O 13ul                
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul               2
 100uM Solexa_2_PE adaptor  0.5ul         0.5ul                 0.5ul               2
 2xQuickLiage buffer         15ul          15ul                  15ul              60
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul               4
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul              10  
 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.

File:20080913 gel cut after adaptor ligation.jpg20080913_gel cut after adaptor ligation

step7. Nick-translation[edit]

 set up the ligation system:
                                          x4              
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul         8ul 
 10mM dNTP                  0.4ul        1.6ul    
 1mg/ml BSA                   2ul         8ul   
 Bst polymerase(8U/ul)        1ul         4ul    
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library[edit]

                                               x4
 Nick-translated DNA          20ul             
 Solexa_PCR_up(10uM)           2ul            8ul  
 Solexa_PCR_lo(10uM)           2ul            8ul
 2xiProof master mix          50ul           200ul  
 50x SYBG                    0.8ul           3.2ul
 ddH2O                      25.2ul         100.8ul
 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20080913 parellel sequencing library of No 1 9.jpg20080913_parellel sequencing library of No_1_9

Qiaquick column purification. Yields:

 No_1(BJ): 17.2ng/ul (104nM) x 30ul
 No_9(Hybrid_2): 18.6ng/ul (112nM) x 30ul
 Send 15ul of each sample to Bing Ren on 09/15/08.

Blunt cloning of sequencing library and sequencing (2008-10-16)[edit]

refer to LabNotes on [2]