After the puzzling results from the last colony screening, I was able to get more info from Tim Green regarding the insert.
The insert was generated via PCR using OsHV-1 ORF 117 primers from this paper:
This should generate a PCR product of ~1300bp. Knowing that, it’s no wonder my previous colony screen didn’t work; I didn’t set the extension time long enough! I increased the extension time to 90s to allow ample time for generating a 1300bp amplicon.
I re-screened the six re-streaked colonies using both the M13 plasmid primers and the ORF117 primers.
Master mix calcs:
2x Apex Red Master PCR Mix: 80uL
M13 forward: 4uL
M13 reverse: 4uL
Added 20uL to each PCR tube.
A miniscule amount of bacteria was collected from each streak with a sterile 10uL pipet tip, which was used to introduce bacteria to the appropriate PCR tube.
95C – 10mins
95C – 15s
55C – 15s
72C – 90s
72C – 10mins
PCR reactions were run on a 1% agarose 1xTBE gel + EtBr.
5uL of O’GeneRuler DNA Ladder Mix was loaded for sizing.
Well, these results are no less confusing than the previous colony screen!
The strong, fuzzy “band” at ~100bp (the lowest band) is likely primer dimers, based on size/intensity. I could potentially redo this and raise the annealing temperature in hopes of eliminating this.
There is a band at ~600bp which I can’t explain.
Finally, a band is also seen at ~1000bp. This is close to the size of the actual coding sequence (CDS) for this OsHV open reading frame (ORF). The ORF contains some extraneous sequence on both ends of the CDS, leading to the ~1300bp length.
There is a faint, yet defined, band at ~4000bp. Coincidentally, this is very close to the size of the empty plasmid (pCR2.1 is 3.9kb). It could be possible that the band that’s present is actually just the plasmid (although, it hasn’t/shouldn’t be linearized) and not an actual PCR product.
Overall, both results are confusing. I’ll just go ahead and sequence one of the colonies using the M13 primers and see what’s there.