Kun:LabNotes/ExonomeSeq/2007-12-12: Difference between revisions

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Ethanol precipitation, resuspend in 190ul dH2O.
Ethanol precipitation, resuspend in 190ul dH2O.
   [[Image:2007-12-13-E55kv1-lib-PCR.png|400px]]
   [[Image:2007-12-13-E55kv1-lib-PCR.png|400px]]
  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.
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.
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.

Latest revision as of 02:26, 14 December 2007

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.