User:Tara K. Luckau/Notebook/Team ConGen/2011/07/11

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The Next few PCRs

  • since last frag data was so confusing, need to frag uniplexes to confirm migration of each locus
  • multiplexing several markers will take lots of time, so try multiplexing by color, then mixing all colors together after PCRing
since splitting into smaller multiplex, can refine reaction conditions
here is the gradient summary from 13 June, sorted by fluorophore
* indicates chosen conditions:
for Blues, Buffer E @ 55°C
for Green and Yellow, Buffer D @ 64°C)
20110711 GradientByFluorophore.jpg
  • also get frag data on a subsample of SCOCs - 5 samples from each road cluster from each site (2 clusters on same side of road, 1 cluster from opposite side = 15 samples per site)

Multiplex Blue PCR

  • Uniplex of Scun2, Scun10, Scun22
  • Multiplex of Scun2 + Scun10 + Scun22

  • 20110711 PCR1.jpg

Multiplex Green and Yellow PCR

  • Uniplex of Scun3, Scun11
  • Multiplex of Scun3 + Scun11
  • Uniplex of Scun9, Scun15
  • Multiplex of Scun9 + Scun15

  • 20110711 PCR2.jpg

Multiplex Gel

  • Did not run gel on all samples, only a subset, so could fit on one row of a gel

  • Uniplexes all worked well
  • Multiplex Blue doesn't have Scun22 (consistent with past multiplexing attempts)
  • Multiplex Green seemed to fail
  • Multiplex Yellow only has one faint amplicon near where Scun15 would fall

  • submit all to frag, anyway

  • 20110711 Gel12.jpg