Hello,
My labmates designed primers for a Gibson assembly experiment. They did the way they usually do, making a final plasmid and using benchling to design the primers.
As a result of the experiment, the plasmid digestion didn't show expected size bands. The sizes, however, would match if the insert was in a reverse orientation.
I cannot find online any people experiencing this. So I am trying to ask here.
We noticed that the insert in the bacteria genome is in the reverse complementary orientation, from WGS data. So I am wondering if this could be the problem. But after trying to redesign primers with that info, the primers seem to be the same as we ordered previously.
If you have any ideas, please let me know.
Thank you
I think the most likely explanations are: 3' ends of the forward/reverse primers specific to the insert are reversed from what was intended, someone made a mistake on the 5' homology arms and swapped them, or the plasmid region they're assembling into is actually in the opposite orientation of what they think it is.
If you give me a Genbank sequence of the intended plasmid, what strain the insert is from, what the backbone plasmid is, and the sequences of the actual primers ordered, I can take a look at it to try to figure it out.
Hello, Thank you for your answer. I don't think I can give details as my lab is quite strict on confidentiality. But the plasmid is a pCas Plasmid #62225 (addgene)
The insert I can only say in the genome the WGS sequence showed to be in reverse orientation.
We checked the primers and they seem okay.
Primers:
Vector FWD (1)
GTTAAagtatattttagatgaagattatttcttaatct
Vector REV (1)
AACATttttgcctcctaaaataaaaagttt
insert FWD (2)
ttttattttaggaggcaaaaATGTT
insert REV (2)
atcttcatctaaaatatactTTAA
The lower case is the pCas sequences and uppercase are few nucleotides of the insert (from ATG to TAA)
Sorry for removing some information but I hope it can help figuring out.
Thank you
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