Matt:LabNotes/2013-5-11: Difference between revisions

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*20 gap first round amplicon -> AP1V4 primers
*20 gap first round amplicon -> AP1V4 primers
*First round amplicons needed to be diluted to 10nM so made 12ul dilutions of each
*First round amplicons needed to be diluted to 10nM so made 12ul dilutions of each
   0 gap: 12ul x 10nM = 1.07ul * 112nM -> 1.07ul first round amplicon + 10.93 H20
   0 gap: 12ul x 10nM = 1.07ul * 112nM -> 1.071ul first round amplicon + 10.929 H20
   20 gap: 12ul * 10nM = 0.98ul * 123nM -> 0.98ul first round amplicon + 11.02 H2O
   20 gap: 12ul * 10nM = 0.98ul * 123nM -> 0.976ul first round amplicon + 11.024 H2O


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Revision as of 21:16, 11 May 2013

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

Expansion PCR

  • 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