Alice:LabNotes/2009-1-13: Difference between revisions

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==Construct Solexa sequencing library==
==Construct Pair-end Solexa sequencing library==
  (Parallel sequencing library construction)
  (Pair-end sequencing library construction)
  Solexa_1 adaptor and Solexa_2_PE adaptor will be used
  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
  (if it is not parallel sequencing library construction)
  (single-end sequencing library construction)
  Solexa_1 adaptor and Solexa_2 adaptor will be used
  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==
===2nd PCR amplification===
   AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40  
   AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40  
   system setup:                                                  x4           
   system setup:                                                  x4           
Line 15: Line 17:
   AmpR6.3(10uM)                                        2ul        8ul     
   AmpR6.3(10uM)                                        2ul        8ul     
   50x SYBG I                                          0.4ul      1.6ul       
   50x SYBG I                                          0.4ul      1.6ul       
   template(19ng/ul)                                 0.1ul       0.4ul     
   template(19ng/ul)                                   0.1ul     0.4ul     
   Total                                              100ul      400ul     
   Total                                              100ul      400ul     


Line 23: Line 25:
   30ul * 47ng/ul
   30ul * 47ng/ul


==Digestion with MmeI==
===Digestion with MmeI===
   (dUTP 250uM,167.3ng/ul of product will use the system below)       
   (dUTP 250uM,167.3ng/ul of product will use the system below)       
   Total                              20ul       
   Total                              20ul       
Line 41: Line 43:
   37C 2hr
   37C 2hr
    
    
  MinElute column purify. Elute in 22ul EB.
  MinElute column purify. Elute in 11ul EB.


==USER digestion (1/14/09)==
===USER digestion (1/14/09)===
                                  
                                  
   DNA                    10ul         
   DNA                    10ul         
Line 50: Line 52:
   37C 4hr
   37C 4hr


==S1 nuclease digestion==
===S1 nuclease digestion===
                                      
                                      
   10 x S1 nuclease buffer:  2ul     
   10 x S1 nuclease buffer:  2ul     
Line 60: Line 62:
   Minelute cloumn purify. Elute in 16ul H2O.
   Minelute cloumn purify. Elute in 16ul H2O.


==end repair==
===end repair===
                                     positive control
                                     positive control
   Total                  25ul            25ul             
   Total                  25ul            25ul             
Line 72: Line 74:
  Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
  Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.


==adapter ligation (1/15/09)==
===adapter ligation (1/15/09)===
  adaptor constraction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP.  
  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_2_upNH2 and 10ul Solexa_2_lo_noP.  
                           10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP.             
                           10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP.             
Line 92: Line 94:


   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
2D gel result:
[[Image:ZhangLab_2 2009-01-16 14hr 37min.jpg]]
the left most lane is the 25bp ladder,
then cosmic adaptor ligation product, positive control, and negative control


==Nick-translation (1/16/09)==
===Nick-translation (1/19/09)===
    
    
   set up the ligation system:
   set up the ligation system:
 
   Total                      20ul               
   Total                      20ul               
   DNA                        15ul               
   DNA                        15ul               
Line 103: Line 109:
   1mg/ml BSA                  2ul             
   1mg/ml BSA                  2ul             
   Bst polymerase(8U/ul)        1ul             
   Bst polymerase(8U/ul)        1ul             
  two tubes: product with cosmic probe and positive control


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


==PCR of sequencig library==
===PCR of sequencig library===
                                                x3
                                             
   Nick-translated DNA         10ul             
   Nick-translated DNA             10ul             
   Solexa_PCR_up(10uM)           2ul           6ul 
   Solexa_PCR_upH(10uM)             2ul            
   Solexa_PCR_lo(10uM)          2ul            6ul
   Solexa_PCR_PE_loH(10uM)          2ul             
   2xiProof master mix         50ul         150ul 
   2xiProof master mix             50ul          
   50x SYBG                   0.8ul          2.4ul
   50x SYBG                       0.8ul           
   ddH2O                     35.2ul         106.6ul
   ddH2O                         35.2ul      
   98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
 
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:
[[Image:ZhangLab_2 2009-01-19 16hr 14min.jpg]]
the left lane is the positive control,
the middle lane with smear is the cosmic solexa sequencing product,
the right lane is the 25bp ladder.


Purify with Qiaquick column. Elute in 30ul EB.
==Conclusion==
  The solexa sequencing library preparation is successful because the PCR product had the smear at the right size.
  Here are few things to look out for future shot-gun library preparation. One of the most common and important cause of
  unsuccessful product is from the dysfunction of ATP. Since ATP provides energy for most of the biochemical reaction, it is
  advised to always use fresh ATP for each step. Also, it is necessary to include a positive and negative control throughout
  the experiment, because one needs to monitor every step in order to find the cause of any possible mistake.

Latest revision as of 17:46, 9 March 2009

Construct Pair-end Solexa sequencing library[edit]

(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[edit]

 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[edit]

 (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)[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]

                                   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)[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    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:
File:ZhangLab 2 2009-01-16 14hr 37min.jpg
the left most lane is the 25bp ladder,
then cosmic adaptor ligation product, positive control, and negative control

Nick-translation (1/19/09)[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             

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

PCR of sequencig library[edit]

 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:
File:ZhangLab 2 2009-01-19 16hr 14min.jpg
the left lane is the positive control, 
the middle lane with smear is the cosmic solexa sequencing product,
the right lane is the 25bp ladder.

Conclusion[edit]

 The solexa sequencing library preparation is successful because the PCR product had the smear at the right size.
 Here are few things to look out for future shot-gun library preparation. One of the most common and important cause of
 unsuccessful product is from the dysfunction of ATP. Since ATP provides energy for most of the biochemical reaction, it is
 advised to always use fresh ATP for each step. Also, it is necessary to include a positive and negative control throughout
 the experiment, because one needs to monitor every step in order to find the cause of any possible mistake.