Alice:LabNotes/2009-9-15

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Revision as of 21:15, 15 September 2009 by >Jie deng (→‎Digestion with MmeI)
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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

                                   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.
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    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 column. Elute in 20ul H2O.

Nick-translation

 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.

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