Kun:LabNotes/ASE/2007-11-13

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Exp.1 eSNPs capture (cont. on the 2007-11-11 exp.)

Perform PCR on 5ul capturing reactions (100ul for gDNA).

                          x 1        x 12
     template:            5ul         each
     ABI 10x buffer:    4.5ul         54ul
     25mM MgCl2:          3ul         36ul
     10mM dNTP:           1ul         12ul
     100uM AmpF2Sol:   0.15ul        1.8ul
     100uM AmpRSol:    0.15ul        1.8ul
     50X SYBG I:        0.4ul        4.8ul
     iTaq:              0.5ul          6ul
     H2O:              35.3ul        430ul

94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.

  File:2007-11-14-Hues6-eSNP84k-PCR.png

Exp.2 Probe prep. for the CES22k set on two new libraries

Emily resynthesized two libraries for the CES22k set.

  1. CES22k 01783910053 (received Nov. 2007, same sequences as the above library)
  2. CES22k 10783810008 (received Nov. 2007, contains Solexa amplification adaptors)

I'm resuspending the oligos in 100ul nuclease free H2O for the concentration of 100nM.

PCR (with iTaq)

                             x1        x16x2 
        template(100nM)     0.1ul     2x1.6ul    
        10x ABI buffer       10ul       32ul
        25mM MgCl2            6ul       192ul
        10mM dNTP             2ul        64ul
        100uM pAP1V6U       0.3ul       9.6ul
        100uM phosAP2V6     0.3ul       9.6ul
        50X SYBG I          0.8ul      25.6ul
        iTaq                  1ul        32ul
        H2O                  80ul      2560ul

94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Ethanol precipitation, resuspend in 135ul dH2O.

Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 4h, 75C 15min, ethanol precipitation, resuspend in 100ul dH2O.

Dpn II & USER digestion: Add 13.5ul 10X Dpn II buffer, 5ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 10ul Dpn II, 5ul USER, 37C 8h, 75C 10min.