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

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==sequencing library construction for paralleling sequencing ==
==parallel sequencing library construction for No_1(BJ) and No_9(Hybrid_2)==


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 ==
Line 12: Line 12:
   AmpR6.3(10uM)                                        2ul        8ul       
   AmpR6.3(10uM)                                        2ul        8ul       
   50x SYBG I                                          0.4ul      1.6ul       
   50x SYBG I                                          0.4ul      1.6ul       
   template(4ng/ul)                                    0.5ul       2ul      
   template(4ng/ul)                                    0.1ul      0.5ul     
   Total                                              100ul      400ul       
   Total                                              100ul      400ul       


   94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
   94C 3min -> 11 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C


   Bioneer column purification. Yield:
   Bioneer column purification. Yield:
   No_1: 33.2ng/ul x 30ul  
   No_1: 30.5ng/ul x 30ul  
   No_9: 49.4ng/ul x 30ul
   No_9: 44.7ng/ul x 30ul


==Digestion with MmeI==
==Digestion with MmeI==
                                       No_1_2_3_9    x4
                                       No_1_9        x2
   Total                                30ul                   
   Total                                30ul                   
   dUTP_PCR                            20ul         
   dUTP_PCR                            20ul         
   10X NEBuffer 4                      4ul          16            
   10X NEBuffer 4                      4ul          8            
   1mM SAM(fresh)                      4ul          16              
   1mM SAM(fresh)                      4ul          8              
   2U/ul Mme I                          8ul          32                
   2U/ul Mme I                          8ul          16                
   ddH2O                                4ul          16
   ddH2O                                4ul          8
    
    
   1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
   1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
   37C 2h. MinEulte column purification. Elute in 12ul EB.
   37C 2h. MinEulte column purification. Elute in 12ul EB.
[[Image:20080912_dUTP_PCR and MmeI of No_1_9.jpg]]20080912_dUTP_PCR and MmeI of No_1_9


==step3. USER digestion==
==step3. USER digestion==
Line 42: Line 44:


==step4. S1 nuclease digestion==
==step4. S1 nuclease digestion==
                             No1_2_3_9      x4
                             No1_9          x2
   10 x S1 nuclease buffer:  2ul            8ul
   10 x S1 nuclease buffer:  2ul            4ul
   DNA after USER digestion: 13ul            13ul
   DNA after USER digestion: 13ul            13ul
   S1 nuclease (10U/ul):      1ul            4ul
   S1 nuclease (10U/ul):      1ul            2ul
   ddH2O                      4ul            16ul
   ddH2O                      4ul            8ul


   37C 10mins.
   37C 10mins.
Line 52: Line 54:


==step5. end repair==
==step5. end repair==
                       No1_2_3_9         positive control               x5
                       No1_9         positive control                   x3
   Total                  25ul          H2O 13ul             
   Total                  25ul          H2O 13ul             
   DNA                    15ul            2ul(20080801_spacer_107bp)             
   DNA                    15ul            2ul(20080801_spacer_107bp)             
   dNTP                  2.5ul          2.5ul                          12.5  
   dNTP                  2.5ul          2.5ul                          7.5  
   dATP                  2.5ul          2.5ul                          12.5
   dATP                  2.5ul          2.5ul                          7.5
   10xendrepair buffer  2.5ul          2.5ul                          12.5  
   10xendrepair buffer  2.5ul          2.5ul                          7.5  
   enzyme                0.5ul          0.5ul                           2.5
   enzyme                0.5ul          0.5ul                           1.5
   extra ATP(10mM)      2.5ul          2.5ul                          12.5
   extra ATP(10mM)      2.5ul          2.5ul                          7.5


   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
   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==
==step6. adapter ligation==
  adaptor constraction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 95C 5mins -> 65C 30mins -> 4C.
                            
                            
                           No1_2_3_9    positive control    negative control       x6
                           No1_9    positive control    negative control         x4
   DNA                        13ul          13ul            ddH2O 13ul             48 
   DNA                        13ul          13ul            ddH2O 13ul              
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul              3
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul              2
   100uM Solexa_2 adaptor     0.5ul        0.5ul                0.5ul              3
   100uM Solexa_2_PE adaptor 0.5ul        0.5ul                0.5ul              2
   2xQuickLiage buffer        15ul          15ul                  15ul              90
   2xQuickLiage buffer        15ul          15ul                  15ul              60
   QuickLigase enzyme(NEB)      1ul          1ul                  1ul              6
   QuickLigase enzyme(NEB)      1ul          1ul                  1ul              4
   extra ATP(10mM)            2.5ul        2.5ul                2.5ul              15  
   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.
   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.
   TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.
[[Image:20080913_gel cut after adaptor ligation.jpg]]20080913_gel cut after adaptor ligation


==step7. Nick-translation==
==step7. Nick-translation==
    
    
   set up the ligation system:
   set up the ligation system:
                                           x6              
                                           x4              
   Total                      20ul               
   Total                      20ul               
   DNA                        15ul               
   DNA                        15ul               
   10x ThermoPol buffer        2ul        12ul
   10x ThermoPol buffer        2ul        8ul
   10mM dNTP                  0.4ul        2.4ul    
   10mM dNTP                  0.4ul        1.6ul    
   1mg/ml BSA                  2ul        12ul    
   1mg/ml BSA                  2ul        8ul    
   Bst polymerase(8U/ul)        1ul         6ul    
   Bst polymerase(8U/ul)        1ul         4ul    


   65C for 25 minutes -> keep on ice.
   65C for 25 minutes -> keep on ice.


==step8. PCR of sequencig library==
==step8. PCR of sequencig library==
                                                 x6
                                                 x4
   Nick-translated DNA          20ul             
   Nick-translated DNA          20ul             
   Solexa_PCR_up(10uM)          2ul            12ul  
   Solexa_PCR_up(10uM)          2ul            8ul  
   Solexa_PCR_lo(10uM)          2ul            12ul
   Solexa_PCR_lo(10uM)          2ul            8ul
   2xiProof master mix          50ul          300ul  
   2xiProof master mix          50ul          200ul  
   50x SYBG                    0.8ul          4.8ul
   50x SYBG                    0.8ul          3.2ul
   ddH2O                      25.2ul        151.2ul
   ddH2O                      25.2ul        100.8ul
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
[[Image:20080913_parellel sequencing library of No_1_9.jpg]]20080913_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)==
refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-10-16]

Latest revision as of 16:54, 16 October 2008

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]