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

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  capture reaction setup can be found under LabNotes 8-15-09 and 8-16-09
  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)
  samples to be used: D3(6&7 subsets of NA12878), DF6-9-9, foreskin, ips(PGP1-ips), Cvi(CV-ips), CVf(CV-fibroblast)
Check LabNotes 9-14-09 for the details on re-amplification PCR and the ratio to pool all the products together


==Procedure==
==Procedure==
Line 65: Line 66:


   37C 10mins.
   37C 10mins.
   Minelute cloumn purify. Elute in 20ul H2O.
   Minelute cloumn purify. Elute in 16ul H2O.


===end repair===
===end repair===
Line 79: Line 80:
  Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O.
  Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O.
  PAGE size selection
  PAGE size selection
[[Image:ZhangLab_2 2009-09-16 12hr 47min.jpg]]  [[Image:ZhangLab_2 2009-09-16 12hr 48min.jpg]]


===adapter ligation===
===adapter ligation===
Line 87: Line 89:


   
   
  total                          30ul   positive control    negative control        
  total                          30ul       
   DNA                            13ul         13ul            ddH2O 13ul           
   DNA                            13ul            
   100uM Solexa_1 adaptor        0.5ul        0.5ul                0.5ul
   100uM Solexa_1 adaptor         1ul        
   100uM Solexa_2 adaptor     0.5ul         0.5ul                0.5ul
   100uM Solexa_2 adaptor         1ul          
   2xQuickLiage buffer            15ul          15ul                  15ul
   2xQuickLiage buffer            15ul           
   QuickLigase enzyme(NEB)        1ul          1ul                  1ul
   QuickLigase enzyme(NEB)        1ul           
   extra ATP(10mM)              2.5ul         2.5ul                2.5ul
   extra ATP(10mM)              2.5ul      
   
   
   
   
   Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 20ul H2O.
   Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 10ul H2O.


===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===
                                                
                                                
   Nick-translated DNA             10ul              
   Nick-translated DNA               6ul              
   Solexa_PCR_up(100uM)            0.2ul             
   Solexa_PCR_up(100uM)            0.2ul             
   Solexa_PCR_lo(100uM)            0.2ul             
   Solexa_PCR_lo(100uM)            0.2ul             
   2xiProof master mix              50ul           
   2xiProof master mix              50ul           
   50x SYBG                        0.8ul          
   50x SYBG                        0.2ul          
   ddH2O                         35.2ul        
   ddH2O                           44ul        
    
    
   98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
   98C 30sec -> 7-8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
PAGE size selection
  PAGE size selection 175-225bp
  [[Image:ZhangLab_2 2009-09-17 16hr 21min-1.jpg]]
 
===QPCR===
template                  1ul     
Phusion 2x master mix    25ul
Syb FP5(100uM)            0.2ul
Syb RP7(100uM)            0.2ul
50X SYBR green            0.2ul
H2O                      24ul
 
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20 sec)X21 -> 72C 3min -> 15C
 
results:
DF6-9-9:        12.3nM
foreskin:        5nM
CV-ips:          26.3nM
CV-fibroblast:  10nM

Latest revision as of 20:57, 18 September 2009

Single-end solexa sequencing library[edit]

Materials[edit]

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)
Check LabNotes 9-14-09 for the details on re-amplification PCR and the ratio to pool all the products together

Procedure[edit]

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[edit]

 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[edit]

 (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[edit]

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

S1 nuclease digestion[edit]

 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[edit]

 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
File:ZhangLab 2 2009-09-16 12hr 47min.jpg   File:ZhangLab 2 2009-09-16 12hr 48min.jpg

adapter ligation[edit]

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[edit]

 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[edit]

 Nick-translated DNA               6ul             
 Solexa_PCR_up(100uM)            0.2ul             
 Solexa_PCR_lo(100uM)            0.2ul            
 2xiProof master mix              50ul           
 50x SYBG                        0.2ul          
 ddH2O                            44ul        
 
 98C 30sec -> 7-8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
 PAGE size selection 175-225bp
 File:ZhangLab 2 2009-09-17 16hr 21min-1.jpg

QPCR[edit]

template                  1ul      
Phusion 2x master mix     25ul
Syb FP5(100uM)            0.2ul
Syb RP7(100uM)            0.2ul
50X SYBR green            0.2ul
H2O                       24ul
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20 sec)X21 -> 72C 3min -> 15C
results:
DF6-9-9:         12.3nM
foreskin:        5nM
CV-ips:          26.3nM
CV-fibroblast:   10nM