I cloned a fairly large cDNA into a pcDps backbone previously modified with a BsrGI restriction site. I was able to cut the cDNA out of a plasmid with the respective enzyme and inserted it into the vector. I performed a test digest with BsrGI and got the expected product.
However, after performing a test digest with BsrGI and BsaI, I noticed that one band was missing. This was always the lowest band, even after I repeated it with a different enzyme that cut into both the backbone and the construct.
Does anyone have any idea what this could cause because I actually assume that the construct is intact when I remove it from another plasmid?
Thanks for help :)
From what I understand from the question, does the insert also have the BsaI digestion site?
Hi there,
You may not have cloned the right cDNA. Did you check the construct by sequencing?
How small is the smallest band? There is a minimum amount of DNA needed to be reliably visible on an agarose gel (~ 10 ng for Ethidium bromide). If the smallest band is really small, you need to load more of the digested plasmid to have a chance of seeing it.
You can also try "over-exposing" the gel relative to the larger bands to see if there is a faint, smaller band.
Good luck!
Hello, thank you very much for your help.
My insert contains 3 BsaI restriction sites. I also sequenced my N and C terminus to check if the ligation worked and everything looked good.
The smallest band that disappeared was about 800 bp.
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