Kun:LabNotes/ExonomeSeq/2007-12-12

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Exp.1 Received the Agilent library today, start to make probes[edit]

PCR (with iTaq)

                             x1          x32                       x1      x32   
        E55k1(20nM)         0.1ul       3.2ul     CES22k #4/#5   0.05ul   1.6ul
        10x Sigma buffer     10ul        32ul                      10ul    32ul
        10mM dNTP             2ul        64ul                       2ul    64ul
        100uM pAP1V6U       0.3ul       9.6ul                     0.3ul   9.6ul
        100uM phosAP2V6     0.3ul       9.6ul                     0.3ul   9.6ul
        50X SYBG I          0.8ul      25.6ul                     0.8ul  25.6ul
        iTaq                  1ul        32ul                       1ul    32ul
        H2O                85.5ul      2736ul                     85.5ul 2736ul

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

Ethanol precipitation, resuspend in 190ul dH2O.

 File:2007-12-13-E55kv1-lib-PCR.png
 Gel quantification:
 E55kv1:   17ng/ul x 190ul = 3.23ug
 CES22k#4: 26ng/ul x 190ul = 4.94ug
 CES22k#5: 25ng/ul x 190ul = 4.75ug

Lambda exo digestion: add 20ul 10X lambda exo buffer to 170ul amplicons, 10ul lambda exo, 37C 4h, 75C 15min, ethanol precipitation, resuspend in 160ul dH2O.

Dpn II & USER digestion: Add 20ul 10X Dpn II buffer, 10ul 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 16h, 75C 10min.