Kun:LabNotes/ASE/2007-11-10

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Exp.1 Probe prep. for the CES22k set[edit]

In this round of probe prep, I'm making probes from three different batches of Agilent oligos.

  1. CES22k (from Harvard, has been sequenced)
  2. CES22k 01783910026 (received Oct. 2007, same sequences as the above library)
  3. CES22k 10783810002 (received Oct. 2007, contains Solexa amplification adaptors)

Emily told me tube #2 and #3 should have 10pmol of DNA, so I'm resuspending the oligos in 100ul nuclease free H2O for the concentration of 100nM.

PCR (with iTaq)

                             x1        x16x3 
        template(100nM)     0.1ul     3x1.6ul    
        10x ABI buffer       10ul       480ul
        25mM MgCl2            6ul       288ul
        10mM dNTP             2ul        96ul
        100uM pAP1V6U       0.3ul      14.4ul
        100uM phosAP2V6     0.3ul      14.4ul
        50X SYBG I          0.8ul      38.4ul
        iTaq                  1ul        48ul
        H2O                  80ul      3840ul

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.