I have inserted a 300bp sequence of interest into the PTRV2 vector for use in VIGS and transformed that vector into E.coli, plated on LB Agar w/ 1% Kanamycin. There was growth on the plates, but confirmatory testing with colony PCR showed no amplification for some reason. I re-performed ligation, transformation, and colony PCR and still no amplification.
I've troubleshooted using a longer extension on the off chance the insert was dimerizing/trimerizing, but still no amplification. I routinely use these primers for colony PCR and have not had any issues before. I sent the plasmid off for Sanger sequencing with these same primers and they were unable to determine the sequence. All signs seem to point to the primers being the issues, but how can this be justified given they've not had any previous issues? How would simply adding a different insert render them ineffective? Is my most logical next step to design new primers for this one instance?
Hello,
Maybe the problem is in your primers. I advise you to check the size of your plasmid by electrophoresis.
Take an empty plasmid that has not received an insert.
Take your plasmid with the insert and perform electrophoresis.
Compare the size of the 2 plasmids (with and without the insert).
The size you get can tell you if the insert is present or not.
Primers go bad over time, especially once they're in solution and subjected to repeated freeze-thaws (or even worse, left out thawed). This happens even quicker if they're in water instead of TE buffer. I agree with the above comment, you need to see if there's any results with a positive control.
In the future if anything looks weird with PCR using an old pair of primers, one of the first things to do is just re-order the primers. A primer pair for colony PCR typically costs less than $20 USD, which is extremely cheap compared to almost everything else.
The easiest thing to do is order new primers and preferably multiple sets just to be sure
Colony PCR might fail due to an overdose of bacteria, you should resuspend the colony to hardly visible turbidity, and then use 1~2ul of the suspension in 20ul PCR reaction.
To much bacteria might bring in PCR inhibitors
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