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)
- 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
Multiplex Green and Yellow PCR
- Uniplex of Scun3, Scun11
- Multiplex of Scun3 + Scun11
- Uniplex of Scun9, Scun15
- Multiplex of Scun9 + Scun15
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
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