Jie:LabNotes/CpgSeq/2009-1-13: Difference between revisions

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   94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
   94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
    
    
   Bioneer column purification. Yield:167ng/ul x60ul
   Qiaquick column purification.  
  Yield:
  BJ: 28.3ng/ul x30ul;
  Hybrid_1: 37.5ng/ul x 30ul;
  Hybrid_2: 39.1ng/ul;
  PGPF1: 30.7ng/ul;
  Hues12: 35.4ng/ul.


==step2. Digestion with MmeI==
==step2. Digestion with MmeI==
                        dUTP 250uM,167.3ng/ul      x2
                                                    x5
   Total                              20ul      
   Total                              30ul      
   DNA                                 6ul          12 
   DNA                                 20ul           
   10X NEBuffer 4                      2ul          4
   10X NEBuffer 4                      4ul          20
   1mM SAM(fresh)                      2ul          4
   1mM SAM(fresh)                      4ul          20
   2U/ul Mme I                          8ul          16
   2U/ul Mme I                          8ul          40
   ddH2O                                2ul          4
   ddH2O                                4ul          20
    
    
   1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
   1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
   37C 2h
   37C 2h
   MinElute column purify. Elute in 22ul EB.
   MinElute column purify. Elute in 11ul EB.


[[Image:20080801_after MmeI.jpg]]20080801_after MmeI
[[Image:2000914_MmeI.jpg]]2000914_MmeI


==step3. USER digestion==
==step3. USER digestion==
                                   x2
                                   x5
   DNA                    10ul     20ul 
   DNA                    10ul        
   USER                    3ul       6ul  
   USER                    3ul     15ul  
   total                  13ul           
   total                  13ul           


37C 12h.
37C 12h.


==step4. S1 nuclease digestion==
==step4. S1 nuclease digestion (2009-01-13)==
                                     x2
                                     x5
   10 x S1 nuclease buffer:  2ul    4ul
   10 x S1 nuclease buffer:  2ul    10ul
   DNA after USER digestion: 13ul   26ul
   DNA after USER digestion: 13ul    
   S1 nuclease (10U/ul):      1ul    2ul
   S1 nuclease (10U/ul):      1ul    5ul
   ddH2O                      4ul    8ul
   ddH2O                      4ul    20ul


   37C 10mins.
   37C 10mins.
   Minelute cloumn purify. Elute in 18ul H2O.
   Minelute cloumn purify. Elute in 16ul H2O.
 
[[Image:20080801_after USER and S1.jpg]]20080801_after USER and S1


==step5. end repair==
==step5. end repair==
                                     positive control
                                     positive control               x6
   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
   dNTP                  2.5ul          2.5ul                       15
   dATP                  2.5ul          2.5ul    
   dATP                  2.5ul          2.5ul                       15
   10xendrepair buffer  2.5ul          2.5ul
   10xendrepair buffer  2.5ul          2.5ul                       15
   enzyme                0.5ul          0.5ul
   enzyme                0.5ul          0.5ul                       3
   extra ATP(10mM)      2.5ul          2.5ul  
   extra ATP(10mM)      2.5ul          2.5ul                       15


   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 14ul H2O.
Line 72: Line 76:
==step6. adapter ligation==
==step6. adapter ligation==
                            
                            
   total                      30ul    positive control    negative control      
   total                      30ul    positive control    negative control   x7 
   DNA                        13ul          13ul            ddH2O 13ul             
   DNA                        13ul          13ul            ddH2O 13ul             
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul         3.5
   100uM Solexa_2 adaptor     0.5ul        0.5ul                0.5ul
   100uM Solexa_2_PE adaptor 0.5ul        0.5ul                0.5ul         3.5
   2xQuickLiage buffer        15ul          15ul                  15ul
   2xQuickLiage buffer        15ul          15ul                  15ul         105
   QuickLigase enzyme(NEB)      1ul          1ul                  1ul
   QuickLigase enzyme(NEB)      1ul          1ul                  1ul           7
   extra ATP(10mM)            2.5ul        2.5ul                2.5ul
   extra ATP(10mM)            2.5ul        2.5ul                2.5ul         17.5


   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.
[[Image: 20080801_after endrepair and ligation.jpg]]


   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
Line 89: Line 91:
    
    
   set up the ligation system:
   set up the ligation system:
 
                                      x7
   Total                      20ul               
   Total                      20ul               
   DNA                        15ul               
   DNA                        15ul               
   10x ThermoPol buffer        2ul        
   10x ThermoPol buffer        2ul     14     
   10mM dNTP                  0.4ul          
   10mM dNTP                  0.4ul   2.8     
   1mg/ml BSA                  2ul          
   1mg/ml BSA                  2ul     14     
   Bst polymerase(8U/ul)        1ul            
   Bst polymerase(8U/ul)        1ul     7     


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


==step8. PCR of sequencig library==
==step8. PCR of sequencig library==
                                                 x3
                                                 x7
   Nick-translated DNA          10ul              
   Nick-translated DNA          20ul              
   Solexa_PCR_up(10uM)           2ul            6ul  
   Solexa_PCR_upH(10uM)         2ul            14ul  
   Solexa_PCR_lo(10uM)           2ul            6ul
   Solexa_PCR_PE_loH(10uM)       2ul            14ul
   2xiProof master mix          50ul          150ul  
   2xiProof master mix          50ul          1050ul  
   50x SYBG                    0.8ul         2.4ul
   50x SYBG                    0.8ul           5.6ul
   ddH2O                      35.2ul        106.6ul
   ddH2O                      25.2ul        176.4ul
   98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
   98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.


[[Image:20080803_sequencing library.jpg]]20080803_sequencing library
[[Image:20090120_sequencing library.jpg]]20090120_sequencing library


Purify with Qiaquick column. Elute in 30ul EB.
  Purify with Qiaquick column. Elute in 30ul EB.
yield: 22.9ng/ul(173nM)
  yield:  
  No_1 BJ: 13.7ng/ul(75.4nM) x 30ul
  No_8 Hybrid_1: 15.9ng/ul(87.6nM) x 30ul
  No_9 Hybrid_2: 16.1ng/ul(88.7nM) x 30ul
  No_17 PGPF1: 19.3ng/ul(106nM) x 30ul
  No_18 HUES12: 13.6ng/ul(74.9nM) x 30ul

Latest revision as of 00:30, 31 January 2009

sequencing library construction (2009-01-13) of BJ, Hybrid_1, Hybrid_2, PGP1F, Hues12[edit]

 No1: BJ (4.8ng/ul, 09/01/08);
 NO8: Hybrid_1 (4ng/ul, 08/04/08);
 No9: Hybrid_2 (2.8ng/ul, 09/01/08);
 No17: PGPF1 (7ng/ul, 10/10/08);
 No18: Hues12 (6ng/ul, 11/04/08).

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

 reaction system                                                x4         x5 
 H2O                                                43.2ul     172.8ul    864
 2x Master mix                                        50ul      200ul    1000
 dUTP(1mM)                                             2ul        8ul      40
 AmpF6.3(10uM)                                         2ul        8ul      40
 AmpR6.3(10uM)                                         2ul        8ul      40
 50x SYBG I                                          0.4ul      1.6ul       8
 template(18.4ng/ul)                                 0.1ul      0.4ul       2
 Total                                               100ul      400ul     2000
 94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Qiaquick column purification. 
 Yield:
 BJ: 28.3ng/ul x30ul;
 Hybrid_1: 37.5ng/ul x 30ul;
 Hybrid_2: 39.1ng/ul;
 PGPF1: 30.7ng/ul;
 Hues12: 35.4ng/ul.

step2. Digestion with MmeI[edit]

                                                   x5 
 Total                               30ul      
 DNA                                 20ul            
 10X NEBuffer 4                       4ul          20 
 1mM SAM(fresh)                       4ul          20
 2U/ul Mme I                          8ul          40 
 ddH2O                                4ul          20 
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
 MinElute column purify. Elute in 11ul EB.

File:2000914 MmeI.jpg2000914_MmeI

step3. USER digestion[edit]

                                 x5
 DNA                    10ul         
 USER                    3ul     15ul  
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion (2009-01-13)[edit]

                                   x5
 10 x S1 nuclease buffer:   2ul     10ul
 DNA after USER digestion: 13ul     
 S1 nuclease (10U/ul):      1ul     5ul
 ddH2O                      4ul     20ul
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

step5. end repair[edit]

                                   positive control                x6
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul                       15
 dATP                  2.5ul           2.5ul                       15
 10xendrepair buffer   2.5ul           2.5ul                       15
 enzyme                0.5ul           0.5ul                        3
 extra ATP(10mM)       2.5ul           2.5ul                       15
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation[edit]

 total                       30ul    positive control     negative control    x7   
 DNA                         13ul          13ul            ddH2O 13ul            
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul         3.5
 100uM Solexa_2_PE adaptor  0.5ul         0.5ul                 0.5ul         3.5
 2xQuickLiage buffer         15ul          15ul                  15ul         105
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul           7
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul         17.5
 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[edit]

 set up the ligation system:
                                      x7
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul     14      
 10mM dNTP                  0.4ul    2.8       
 1mg/ml BSA                   2ul     14       
 Bst polymerase(8U/ul)        1ul      7       
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library[edit]

                                               x7
 Nick-translated DNA          20ul             
 Solexa_PCR_upH(10uM)          2ul            14ul  
 Solexa_PCR_PE_loH(10uM)       2ul            14ul
 2xiProof master mix          50ul          1050ul  
 50x SYBG                    0.8ul           5.6ul
 ddH2O                      25.2ul         176.4ul
 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20090120 sequencing library.jpg20090120_sequencing library

 Purify with Qiaquick column. Elute in 30ul EB.
 yield: 
 No_1 BJ: 13.7ng/ul(75.4nM) x 30ul
 No_8 Hybrid_1: 15.9ng/ul(87.6nM) x 30ul
 No_9 Hybrid_2: 16.1ng/ul(88.7nM) x 30ul
 No_17 PGPF1: 19.3ng/ul(106nM) x 30ul
 No_18 HUES12: 13.6ng/ul(74.9nM) x 30ul