Kun:LabNotes/ExonomeSeq/2008-1-24

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Exp.1 Library construction on the new capturing reaction started on 01/16/2008

Mme I digestion on the 2nd round PCR amplicon

          500bp+ 2nd PCR       all size 2nd PCR
              50ul                   50ul
 32mM SAM    0.5ul                  0.5ul
 Mme I 2U/ul   4ul                    4ul
 37C 1h -> 75C 20min -> 4C hold.
 Purify with Bioneer columns, then PAGE size selection.
 Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis.

USER enzyme digestion

Add 2ul USER enzyme into 18ul of DNA in EB buffer, 37C 1h.

End repair

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

                 USER         
     DNA         19ul        
     10X buffer 2.5ul        
     1mM dNTP   2.5ul         
     Enzyme mix   1ul          
     RT for 30min -> 75C 10min

SolN6 primed synthesis

                       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.

Ligation

                           USER      
      DNA                  10ul              
  50uM Solexa_1             1ul              
  50uM Solexa_2             1ul               
  2X Quick ligation buffer 15ul               
  Quick ligase              1ul                 
  H2O                       2ul                
  mix well -> room temperature for 20min -> 75C 10min
  Purified with MinElute columns.

Nick-translation

  DNA                    15ul
  10X ThermoPol Buffer    2ul
  10mM dNTP             0.4ul
  1mg/ml BSA              2ul
  8U/ul Bst pol           1ul
  65C 25min -> place on ice

PCR

    Nick-translated DNA      5ul       SolN6 primed DNA  5ul
    2X iProof master mix    25ul                        25ul
    100uM Solexa_PCR_up   0.25ul                      0.25ul
    100uM Solexa_PCR_lo   0.25ul                      0.25ul
    H2O                   19.3ul                      19.3ul
    50X SYBG I             0.2ul                       0.2ul
    98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold

Exp.2 Probe prep for Cosmic3760, CpG28k and CES27k

PCR (with NEB Taq master mix)

                                   Cosmic3760              CpG28k            CES27k
                             x1          x25     
        Cosmic3760(20nM)    0.1ul       2.5ul  CpgCES (20nM) 2.5ul            2.5ul
        2x Taq master mix    50ul      1250ul               1250ul           1250ul
        100uM AP1V41U       0.4ul        10ul                 10ul  pAP1V6U    10ul
        100uM pAP2V4        0.4ul        10ul                 10ul  phosAP2V6  10ul
        H2O                49.3ul      1230ul               1230ul           1230ul    

94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Ethanol precipitation.

Resuspended in 170ul dH2O, add 20ul 10X Lambda exo buffer, 10ul 5U/ul lambda exo, 37C 3h -> 90C 10min.

Purify with Qiaquick columns. Yield: Cosmic3760=53ng/ul x 80ul; Cpg28k=33ng/ulx80ul; CES27k=33ng/ul x 80ul.

Added 4ul USER enzyme, 37C 2h.

Add 90ul H2O, 20ul 10X DpnII buffer, 10ul DpnIIV4/5ul DpnIIV4/5ul RE-DpnIIs, 94C 5min -> slowly cool down to 37C -> add 10ul Dpn II -> 37C 2h -> add 5ul USER -> 37C 2h -> 75C 10min -> 4C hold.