I am about to send my plasmid + insert in for sequencing and I was testing out a couple of my plasmid specific primers. The amplicon spans my insert, a tetracycline responsive element (TRE)/minimal CMV promoter and part of the EGFP gene. The amplicon should be around 900bp in length, but when I run the PCR product on a gel it shows up in the 450bp region. For digestion, if I linearize the construct it shows up as the proper size, and if I digest out the insert it shows up as the proper size. However, if I digest out the region that spans my primers, I get a smear on the gel.
My first guess would be that this region forms a strong secondary structure. The tetracycline responsive element is composed of bidirectional nucleotide repeats. If I take just the TRE region and use IDTs hairpin analyzer I get hits with delta G's of -25kcal/mol, Tms of 40c, delta Hs of -530kcal/mol and delta s's of -1680kcal/mol
This encompasses about a 250bp region of the 900bp PCR product. Could this be my problem?
PCR amplification from plasmid - Possible product hairpin formation?
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