Jie:LabNotes/CpgSeq/2009-1-13

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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