Alice:LabNotes/2009-3-13: Difference between revisions

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==Objective==
==Objective==
  1.Construct pair-end solexa sequencing library using PCR-product of probes captured with sheared, short DNA PGP1.
  1.Construct single-end solexa sequencing library using PCR-product of probes captured with sheared, short DNA PGP1.
  2.Compare the MmeI digestion efficiency between established capture method and shaking.
  2.Compare the MmeI digestion efficiency between established capture method and shaking.


Line 58: Line 58:
   50x SYBG I                                          0.4ul      1.6ul       
   50x SYBG I                                          0.4ul      1.6ul       
   template (tube1 / tube2)                      1ul/0.5ul    4ul/2ul
   template (tube1 / tube2)                      1ul/0.5ul    4ul/2ul
                                                               
                                                           
   Total                                              100ul      400ul     
   Total                                              100ul      400ul     
   
   
Line 93: Line 93:


  MinElute column purify. Elute in 11ul EB.
  MinElute column purify. Elute in 11ul EB.
TBE gel result:
[[Image:ZhangLab_2 2009-03-14 16hr 19min - TBE.jpg]]              [[Image:ZhangLab_2 2009-03-17 10hr 59min-TBU.jpg]]


===USER digestion (1/14/09)===
===USER digestion (1/14/09)===
Line 133: Line 136:
   DNA                            13ul          13ul            ddH2O 13ul             
   DNA                            13ul          13ul            ddH2O 13ul             
   100uM Solexa_1 adaptor        0.5ul        0.5ul                0.5ul
   100uM Solexa_1 adaptor        0.5ul        0.5ul                0.5ul
   100uM Solexa_2_PE adaptor    0.5ul        0.5ul                0.5ul
   100uM Solexa_2 adaptor    0.5ul        0.5ul                0.5ul
   2xQuickLiage buffer            15ul          15ul                  15ul
   2xQuickLiage buffer            15ul          15ul                  15ul
   QuickLigase enzyme(NEB)        1ul          1ul                  1ul
   QuickLigase enzyme(NEB)        1ul          1ul                  1ul
Line 143: Line 146:


   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
   PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
2D gel result:
  TBE gel result:
 
  [[Image:ZhangLab_2 2009-03-18 10hr 45min.jpg]]


===Nick-translation (1/19/09)===
===Nick-translation (1/19/09)===
Line 156: Line 159:
   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.
Line 164: Line 165:
                                                
                                                
   Nick-translated DNA              10ul             
   Nick-translated DNA              10ul             
   Solexa_PCR_upH(10uM)             2ul             
   Solexa_PCR_up(100uM)           0.2ul             
   Solexa_PCR_PE_loH(10uM)           2ul             
   Solexa_PCR_lo(100uM)       0.2ul             
   2xiProof master mix              50ul           
   2xiProof master mix              50ul           
   50x SYBG                        0.8ul           
   50x SYBG                        0.8ul           
Line 171: Line 172:
    
    
  two tubes: product with cosmic probe and positive control
  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.
   98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
[[Image:3-18-09 PCR of solexa library.jpg]]
red line: control
blue line: PGP1-shaked
Green line: PGP1-CTRL
 
Nanodrop esult:
PGP1-CTRL: 48.5ng/ul
PGP1-shaked: 46ng/ul


  Purify with Qiaquick column. Elute in 30ul EB.
  Purify with Qiaquick column. Elute in 30ul EB.
  TBE gel result:
  TBE gel result:
[[Image:ZhangLab_2 2009-03-18 15hr 20min.jpg]]
==Result==
  There is no significant amount of difference between the Mme1 digestion of CTRL set and shaked DNA set. Therefore we cannot say
that one method is preferred over another. Also, the gel image of Mme1 digestion showed 3 bands that suggests incomplete digestion.
A gel selection might be helpful after Mme1 digestion.
==Discussion==
It is very crucial to use TBE gel to do size selection after adapter ligation step. It is because after adapter ligation, the double
stranded DNA will ligate with only one strand of the double stranded solexa adapter, and leaving a gap between the other strand of
the adapter and the captured probe. This is also the reason why a nick translation step is necessary before PCR amplification, because
it is used to seal up the gap. A TBU gel will denature the DNA and causing the unligated strand of adapter to disassociated from the
adapter and the captured probe, thus giving a undesirable product and the experiment fails.

Latest revision as of 21:39, 26 May 2009

Objective[edit]

1.Construct single-end solexa sequencing library using PCR-product of probes captured with sheared, short DNA PGP1.
2.Compare the MmeI digestion efficiency between established capture method and shaking.

Material[edit]

First attempt:
Obtained two tubes from Dr. Zhang: (1) PGP1-CTRL 56ng/ul;  (2) PGP1-shaked 27ng/ul
Dilute tube (1) 10X by adding 9ul of H2O to 1ul of DNA sample.
For tube (2), only very tiny amount of DNA was left in the tube. Took out around 1ul of DNA and add 9ul of H2O. Nanodrop result
gives out final concentration of 6ng/ul.

(1) PGP1-shaked: 6ng/ul
(2) PGP1-CTRL:   5.6ng/ul
Second attempt (3-16-09):

(1) PGP1-shaked: 12ng/ul
(2) PGP1-CTRL: 5.6ng/ul

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

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                                              1ul        4ul     
 Total                                               100ul      400ul     

 94C 3min -> 10 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Qiaquick column purification.
 tube 1 (shaked): 30ul * 257.1ng/ul
 tube 2  (CTRL):  30ul * 194.6ng/ul
Second attempt (3-16-09):
 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 (tube1 / tube2)                       1ul/0.5ul     4ul/2ul
                                                            
 Total                                               100ul      400ul     

 94C 3min -> 10 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 Qiaquick column purification.
 tube 1 (shaked): 30ul * 87.2ng/ul
 tube 2  (CTRL):  30ul * 75.9ng/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) 
   
 First attempt:
 Total                               20ul  
   
 tube 1(257.1ng/ul)                   4ul     tube 2 (194.6ng/ul)       6ul        
 10X NEBuffer 4                       2ul     10X NEBuffer 4            2ul      
 1mM SAM(fresh)                       2ul     1mM SAM(fresh)            2ul   
 2U/ul Mme I                          8ul     2U/ul Mme I               8ul  
 ddH2O                                4ul     ddH2O                     2ul         
 
 Second attempt:
 Total                               40ul  
   
 DNA                                 13ul
 10X NEBuffer 4                       4ul           
 1mM SAM(fresh)                       6ul        
 2U/ul Mme I                          8ul      
 ddH2O                                9ul       

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

TBE gel result:
File:ZhangLab 2 2009-03-14 16hr 19min - TBE.jpg              File:ZhangLab 2 2009-03-17 10hr 59min-TBU.jpg

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 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.
 TBE gel result:
 File:ZhangLab 2 2009-03-18 10hr 45min.jpg

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             
 65C for 25 minutes -> keep on ice.

PCR of sequencig library[edit]

 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        
 
two tubes: product with cosmic probe and positive control
 98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
File:3-18-09 PCR of solexa library.jpg
red line: control
blue line: PGP1-shaked
Green line: PGP1-CTRL
Nanodrop esult:
PGP1-CTRL: 48.5ng/ul
PGP1-shaked: 46ng/ul
Purify with Qiaquick column. Elute in 30ul EB.
TBE gel result:
File:ZhangLab 2 2009-03-18 15hr 20min.jpg

Result[edit]

 There is no significant amount of difference between the Mme1 digestion of CTRL set and shaked DNA set. Therefore we cannot say
that one method is preferred over another. Also, the gel image of Mme1 digestion showed 3 bands that suggests incomplete digestion.
A gel selection might be helpful after Mme1 digestion.

Discussion[edit]

It is very crucial to use TBE gel to do size selection after adapter ligation step. It is because after adapter ligation, the double
stranded DNA will ligate with only one strand of the double stranded solexa adapter, and leaving a gap between the other strand of 
the adapter and the captured probe. This is also the reason why a nick translation step is necessary before PCR amplification, because
it is used to seal up the gap. A TBU gel will denature the DNA and causing the unligated strand of adapter to disassociated from the
adapter and the captured probe, thus giving a undesirable product and the experiment fails.