Alice:LabNotes/2009-9-15: Difference between revisions

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===Nick-translation===
===Nick-translation===
 
         
  set up the ligation system:
   DNA                        10ul                
   10x ThermoPol buffer       1.2ul           
  Total                      20ul             
   10mM dNTP                  0.5ul                 
   DNA                        15ul                
   Bst polymerase(8U/ul)     0.5ul              
   10x ThermoPol buffer         2ul           
   10mM dNTP                  0.4ul         
  1mg/ml BSA                  2ul           
   Bst polymerase(8U/ul)       1ul              


   65C for 25 minutes -> keep on ice.
   65C for 12 minutes


===PCR of sequencig library===
===PCR of sequencig library===

Revision as of 17:57, 17 September 2009

Single-end solexa sequencing library

Materials

use previously captured samples
capture reaction setup can be found under LabNotes 8-15-09 and 8-16-09
samples to be used: D3(6&7 subsets of NA12878), DF6-9-9, foreskin, ips(PGP1-ips), Cvi(CV-ips), CVf(CV-fibroblast)

Procedure

Reagent setup:

(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

PCR re-amplification

 AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 

 system setup:                                                   x4          
 H2O                                                  46ul      184ul  
 2x KAPA Master mix                                   50ul      200ul     
 dUTP(1mM)                                             4ul       16ul      
 AmpF6.3NH2(100uM)                                   0.4ul        8ul      
 AmpR6.3NH2(100uM)                                   0.4ul        8ul     
 template                                            0.5ul        2ul     
 
  95C 30sec -> (95C 3sec -> 60C 30sec) x 3 -> 60C 2min -> 15C hold.
Qiaquick column purification and elute in 30ul EB.
Nanodrop:
D3: 19.2ng/ul         DF: 33.3ng/ul          fs: 17.7ng/ul
ips: 13ng/ul          cvi: 23.7ng/ul         cvf: 12.2ng/ul

Digestion with MmeI

 (dUTP 250uM,167.3ng/ul of product will use 6ul of DNA along with 2ul of 1mM SAM and 8ul of 2U/ul MmeI) 
                                DF & cvi       ips & cvf       D3 & fs 
 DNA                              30ul          30ul           30ul
 10X NEBuffer 4                   5ul           4ul            5ul
 1mM SAM(fresh)                   6ul           3ul            4ul
 2U/ul Mme I                      8ul           3ul            5ul
 ddH2O                            1ul            -             6ul

 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h
MinElute column purify. Elute in 11ul EB.

USER digestion

 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

 Total                  25ul            
 DNA                    15ul           
 dNTP                    5ul          
 10xendrepair buffer   2.5ul          
 enzyme                  1ul          
 ATP(10mM)               5ul        
                                  
 
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O.
PAGE size selection

adapter ligation

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       
 DNA                            13ul             
 100uM Solexa_1 adaptor          1ul        
 100uM Solexa_2 adaptor          1ul         
 2xQuickLiage buffer            15ul          
 QuickLigase enzyme(NEB)         1ul           
 extra ATP(10mM)               2.5ul        


 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 10ul H2O.

Nick-translation

 DNA                         10ul               
 10x ThermoPol buffer       1.2ul          
 10mM dNTP                  0.5ul                  
 Bst polymerase(8U/ul)      0.5ul             
 65C for 12 minutes

PCR of sequencig library

 Nick-translated DNA              10ul             
 Solexa_PCR_up(100uM)            0.2ul             
 Solexa_PCR_lo(100uM)            0.2ul            
 2xiProof master mix              50ul           
 50x SYBG                        0.8ul          
 ddH2O                          35.2ul        
 
 98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
PAGE size selection