Jie:LabNotes/CpgSeq/2008-7-31: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
No edit summary
No edit summary
 
(6 intermediate revisions by 2 users not shown)
Line 29: Line 29:
   37C 2h
   37C 2h
   MinElute column purify. Elute in 22ul EB.
   MinElute column purify. Elute in 22ul EB.
[[Image:20080801_after MmeI.jpg]]20080801_after MmeI


==step3. USER digestion==
==step3. USER digestion==
Line 47: Line 49:
   37C 10mins.
   37C 10mins.
   Minelute cloumn purify. Elute in 18ul H2O.
   Minelute cloumn purify. Elute in 18ul H2O.
[[Image:20080801_after USER and S1.jpg]]20080801_after USER and S1


==step5. end repair==
==step5. end repair==
                                     positive control
                                     positive control
   Total                  25ul          H2O 13ul             
   Total                  25ul          H2O 13ul             
   DNA                    15ul            2ul(20080801_sapcer_107bp)             
   DNA                    15ul            2ul(20080801_spacer_107bp)             
   dNTP                  2.5ul          2.5ul
   dNTP                  2.5ul          2.5ul
   dATP                  2.5ul          2.5ul       
   dATP                  2.5ul          2.5ul       
Line 70: Line 74:
   extra ATP(10mM)            2.5ul        2.5ul                2.5ul
   extra ATP(10mM)            2.5ul        2.5ul                2.5ul


   Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.
   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.


==step7. Nick-translation==
==step7. Nick-translation==
Line 91: Line 96:
                                                 x3
                                                 x3
   Nick-translated DNA          10ul             
   Nick-translated DNA          10ul             
   Solexa_PCR_up(10uM)          1ul           3ul  
   Solexa_PCR_up(10uM)          2ul           6ul  
   Solexa_PCR_lo(10uM)          1ul           3ul
   Solexa_PCR_lo(10uM)          2ul           6ul
   2xiProof master mix          25ul          75ul  
   2xiProof master mix          50ul          150ul  
   50x SYBG                    0.4ul         1.2ul
   50x SYBG                    0.8ul         2.4ul
   ddH2O                      12.6ul         37.8ul
   ddH2O                      35.2ul         106.6ul
   98C 30sec -> 13 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


Purify with Qiaquick column. Elute in 30ul EB.
Purify with Qiaquick column. Elute in 30ul EB.
dUTP250uM: 12.8ng/ul
yield: 22.9ng/ul(173nM)
dUTP100uM: 22.5ng/ul

Latest revision as of 16:57, 4 August 2008

sequencing library construction[edit]

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

 reaction system                                                x4         x3 
 H2O                                                43.2ul     172.8ul  518.4
 2x Master mix                                        50ul      200ul     600
 dUTP(1mM)                                             2ul        8ul      24
 AmpF6.3(10uM)                                         2ul        8ul      24
 AmpR6.3(10uM)                                         2ul        8ul      24
 50x SYBG I                                          0.4ul      1.6ul     4.8
 template(18.4ng/ul)                                 0.1ul      0.4ul     1.2
 Total                                               100ul      400ul     2400
 94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Bioneer column purification. Yield:167ng/ul x60ul

step2. Digestion with MmeI[edit]

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

File:20080801 after MmeI.jpg20080801_after MmeI

step3. USER digestion[edit]

                                 x2
 DNA                    10ul      20ul   
 USER                    3ul       6ul  
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion[edit]

                                   x2
 10 x S1 nuclease buffer:   2ul     4ul
 DNA after USER digestion: 13ul    26ul
 S1 nuclease (10U/ul):      1ul     2ul
 ddH2O                      4ul     8ul
 37C 10mins.
 Minelute cloumn purify. Elute in 18ul H2O.

File:20080801 after USER and S1.jpg20080801_after USER and S1

step5. end repair[edit]

                                   positive control
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul
 dATP                  2.5ul           2.5ul      
 10xendrepair buffer   2.5ul           2.5ul  
 enzyme                0.5ul           0.5ul
 extra ATP(10mM)       2.5ul           2.5ul 
 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       
 DNA                         13ul          13ul            ddH2O 13ul            
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 0.5ul
 2xQuickLiage buffer         15ul          15ul                  15ul
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul
 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O.

File:20080801 after endrepair and ligation.jpg

 PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.

step7. Nick-translation[edit]

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

step8. PCR of sequencig library[edit]

                                               x3
 Nick-translated DNA          10ul             
 Solexa_PCR_up(10uM)           2ul            6ul  
 Solexa_PCR_lo(10uM)           2ul            6ul
 2xiProof master mix          50ul          150ul  
 50x SYBG                    0.8ul          2.4ul
 ddH2O                      35.2ul         106.6ul
 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20080803 sequencing library.jpg20080803_sequencing library

Purify with Qiaquick column. Elute in 30ul EB. yield: 22.9ng/ul(173nM)