Matt:LabNotes/2013-5-11

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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-10

Production PCR[edit]

  • Made 2 Master mixes (for 0 gap and 20 gap probes)
  • Each MM is for 46 well (half a 96 well plate)
  • 0 gap first round amplicon -> AP1V6 primers
  • 20 gap first round amplicon -> AP1V4 primers
  • First round amplicons needed to be diluted to 10nM so made 12ul dilutions of each
 0 gap: 12ul x 10nM = 1.07ul * 112nM -> 1.071ul first round amplicon + 10.929 H20
 20 gap: 12ul * 10nM = 0.98ul * 123nM -> 0.976ul first round amplicon + 11.024 H2O
Components 1x rxn (ul) 50x rxn (ul)
First round amplicon (10nM) 0.2 10
2x Kapa SYBR MM 50 2500
Forward primer (100uM) 0.4 20
Reverse primer (100uM) 0.4 20
H2O 49 2450
Total 100 5000

98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 16 -> 72C 2min -> 15C hold

  • Stopped after 15 runs

File:051113 ProductionPCR Agi26k.jpg

  • Lower fluorescence samples are due to wells with less than 100ul (even with making 4 extra mastermix samples in previous step, wasn't able to pipette a full 100ul in the last 1-3 wells)

EtOH Precipitation[edit]

  • 6 15-ml tubes (with 8 wells of PCR product each) for 0gap and 6 tubes for 20gap
    • 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 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
  • Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-12