Alice:LabNotes/2009-3-13

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Revision as of 16:57, 13 March 2009 by KunZhang (talk | contribs) (New page: ==Construct Pair-end Solexa sequencing library== (Pair-end sequencing library construction) Solexa_1 adaptor and Solexa_2_PE adaptor will be used for PCR at the last step use these prim...)
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Construct Pair-end Solexa sequencing library

(Pair-end sequencing library construction)
Solexa_1 adaptor and Solexa_2_PE adaptor will be used
for PCR at the last step use these primers:  Solexa_PCR_PE_loH & Solexa_PCR_upH

(single-end sequencing library construction)
Solexa_1 adaptor and Solexa_2 adaptor will be used
for PCR at the last step use these primers:  Solexa_PCR_up Solexa_PCR_lo

2nd PCR amplification

 AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 
 system setup:                                                   x4          
 H2O                                                43.2ul     172.8ul  
 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(19ng/ul)                                   0.1ul      0.4ul     
 Total                                               100ul      400ul     
 94C 3min -> 9 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 
 Qiaquick column purification.
 30ul * 47ng/ul

Digestion with MmeI

 (dUTP 250uM,167.3ng/ul of product will use the system below)      
 Total                               20ul      
 DNA                                  6ul            
 10X NEBuffer 4                       2ul           
 1mM SAM(fresh)                       2ul          
 2U/ul Mme I                          8ul          
 ddH2O                                2ul          
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
 however, since the concentration I obtained from previous is 47ng/ul, therefore the following is added:
 DNA              14ul
 SAM(1mM)          4ul
 NEBuffer 4        2ul
 37C 2hr
 
MinElute column purify. Elute in 11ul EB.

USER digestion (1/14/09)

 DNA                    10ul         
 USER                    3ul     
 total                  13ul           
 37C 4hr

S1 nuclease digestion

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

end repair

                                   positive control
 Total                  25ul            25ul            
 DNA                    15ul             2ul (obtained from Ida: 20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul
 10xendrepair buffer   2.5ul           2.5ul  
 enzyme                0.5ul           0.5ul
 ATP(10mM)               5ul             5ul 
                                  H2O   13ul
 
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

adapter ligation (1/15/09)

adaptor construction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 
                          10ul Solexa_2_upNH2 and 10ul Solexa_2_lo_noP. 
                          10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP.             
95C 5mins -> 65C 30mins -> 4C.


total                           30ul    positive control     negative control       
 DNA                            13ul          13ul            ddH2O 13ul            
 100uM Solexa_1 adaptor        0.5ul         0.5ul                 0.5ul
 100uM Solexa_2_PE 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 column. Elute in 20ul H2O.

 PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
2D gel result:


Nick-translation (1/19/09)

 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             

 two tubes: product with cosmic probe and positive control
 65C for 25 minutes -> keep on ice.

PCR of sequencig library

 Nick-translated DNA              10ul             
 Solexa_PCR_upH(10uM)              2ul             
 Solexa_PCR_PE_loH(10uM)           2ul            
 2xiProof master mix              50ul           
 50x SYBG                        0.8ul          
 ddH2O                          35.2ul        
 
two tubes: product with cosmic probe and positive control
 98C 30sec -> 17 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
Purify with Qiaquick column. Elute in 30ul EB.
TBE gel result: