Kun:LabNotes/ExonomeSeq/2008-1-30

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Exp.1 Library construction on the Cosmic3760 exons from NA10835 and NA20431 (01/27/2008)[edit]

Mme I digestion on the PCR amplicon[edit]

 Mix pooled 50ul amplicons from two tubes, add 1ul 32mM SAM, 4ul Mme I.
 37C 2h -> 75C 20min.
 PAGE size selection of 50ul digestions.
 Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis.

USER enzyme digestion[edit]

Add 1ul USER enzyme into 10ul of DNA in ddH2O -> 37C 10min.

End repair[edit]

Set up end repair reaction (with NEB quick blunting kit)

                 USER         
     DNA         10ul        
     10X buffer 1.5ul        
     1mM dNTP   1.5ul         
     Enzyme mix 0.5ul
     H2O        1.5ul          
     RT for 30min -> 75C 10min

Ligation[edit]

      DNA                  15ul              
  50uM Solexa_1             2ul              
  50uM Solexa_2             2ul               
  2X Quick ligation buffer 20ul               
  Quick ligase              1ul
  mix well -> room temperature for 20min 
  Purified with MinElute columns.

Nick-translation[edit]

  DNA                    10ul
  10X ThermoPol Buffer  1.2ul
  10mM dNTP             0.3ul
  8U/ul Bst pol         0.5ul
  65C 25min -> place on ice

SolN6 primed synthesis[edit]

                       SolF/Rn6     
     DNA                 10ul      
     10X Klenow buffer    5ul     
     100uM SolFn6         1ul
     10mM dNTP            2ul      
     H2O                 30ul      

94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> Clean up the reaction with G25 column, then add 6 ul 10X buffer, 2ul dNTp, 1ul 100uM SolRn6-> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo-, 1ul T4 DNA pol-> 16C 10min -> 37C 10min -> purified with MinElute columns, eluted with 10ul EB.

PCR[edit]

    Nick-translated DNA      5ul       SolN6 primed DNA  5ul
    2X iProof master mix    50ul                        50ul
    100uM Solexa_PCR_up   0.25ul                      0.25ul
    100uM Solexa_PCR_lo   0.25ul                      0.25ul
    H2O                     44ul                        44ul
    50X SYBG I             0.4ul                       0.4ul
    98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
 File:2008-02-01-SolLib-PCR.png
 
 
 I decided to do PAGE purification of the remaining 10ul PCR templates, then do PCR.
 File:2008-02-01-PAGE.png
 After shearing the gel, I directly added 100ul 1X iProof mix with 0.2X SYBR Green I but no primer.
 Incubate at thermal cycler at 37C for 1h, then start thermal cycling with same protocol.
 However, I realized after 15 cycles that I forgot to added the primers. 
 So I took the tubes out, add Solexa_PCR_lo/up primers (0.4uM), and repeat 18 cycles of thermal cycling.
 I took tube A/B out, and did 5 more cycles on C/D.
 PAGE purification on the A/B amplicons from PAGE purified templates, and C/D amplicons from unpurified templates.
 The amplification A/B didn't work.
 File:2008-02-01-SolLib-PCR-PAGE.png