Matt:LabNotes/2014-7-17

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Revision as of 01:32, 18 July 2014 by >Mzcai
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Agi26k0gap Probe Production

Production PCR

  • Make Master Mix for 46 well (6 strips)
  • 0 gap first round amplicon -> V6 primers
Components 1x rxn (ul) 50x rxn (ul)
First round amplicon (10nM) 0.2 10
2x Kapa SYBR MM 50 2500
100uM pAP1V6U 0.4 20
100uM AP2V6 0.4 20
H2O 49 2450
Total 100 5000

98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold

File:2014-07-17 Agi26k0gapProductionPCR.JPG

EtOH Precipitation

  • 6 5-ml tubes (with 8 wells of PCR product each) for 0gap
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 1hr
  • Centrifuged at 3000rpm at 4C for overnight