Kun:LabNotes/ASE/2007-10-18

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Exp. 1 Perform PCR on CES22k and eSNP84k libraries to make MIP probes.[edit]

Reaction CES22k(100nM):

                               x 8
    Template        0.2ul     1.6ul
    10X buffer       10ul      80ul
    10mM dNTP         2ul      16ul
    100uM pAP1V6U   0.4ul     3.2ul
    100uM phosAp2V6 0.4ul     3.2ul
    JumpStart Taq     2ul      16ul
    H2O              85ul     680ul

Reactions eSNP84k set1-5 (100nM):

                              x 8 x 5 
    Template        0.2ul      1.6ul x 5 
    10X buffer       10ul       80ul x 5 
    10mM dNTP         2ul       16ul x 5
    100uM AP1V41U   0.4ul      3.2ul x 5
    100uM Ap2V4     0.4ul      3.2ul x 5
    JumpStart Taq     2ul       16ul x 5
    H2O              85ul      680ul x 5

94C3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 4C hold

  File:2007-10-19-AgilentLib-PCR.png
  Estimated yield:
  CES22k:  12.5ng/ul
  eSNP84k1: 11.5ng/ul
  eSNP84k2: 13ng/ul
  eSNP84k3: 8.5ng/ul
  eSNP84k4: 12.75ng/ul
  eSNP84k5: 15.25ng/ul

Ethanol Precipitation, 400ul reactions per tube, two tubes per sample. Resuspended 50ul dH2O, measured concentration with Nanodrop.

          NaOAC, pH=5  NaOAC, pH=7
  CES22k:   215ng/ul    246ng/ul
  eSNP84k1: 216ng/ul    184ng/ul
  eSNP84k2: 184ng/ul    289ng/ul
  eSNP84k3: 157ng/ul    290ng/ul
  eSNP84k4: 142ng/ul    256ng/ul
  eSNP84k5: 163ng/ul    221ng/ul