I am trying to clone a fusion PCR product in pcDNA3 vector. After double RE digestion, and direct ligation with the vector, bacteria transformation seems to be working. There is no colonies on the control plate without the insert, while there are colonies in the plate with the insert. However, when I ran my miniprep products after digesting with the two REs again, I see only one band at the size of my plasmid vector and not a recombinant one. It is baffling that there were colonies so there should have been recombinant plasmids but the band size after running it on gel does not match.
What was the control plate you did, vector alone or vector plus ligase? It should have been the latter (or both). However before worrying too much, I would suggest just screening a bunch more colonies. Also, are you getting no insert, or do you have an insert but it is the wrong size?
Michael J. Benedik Hello Michael, Thank you for your response. My control plate had vector with ligase. I screened 6 colonies, and all did not have the expected bands. After digesting my mini prep with REs, I am expected two bands ( one for the vector, and one for my insert). However, I only get one band the size of my vector. I am confused as there were colonies on my plate, yet there doesn't seem to be recombinant plasmids. I had double digested the vector with two different REs so I believe the vector couldn't have relegated.
6 is not very many colonies. Try screening a lot more, maybe 24 or so, to see if any have inserts before you give up.
The vector can relegate if one of the enzymes didn't digest completely so even if only a tiny fraction of the vector population was only singly digested you can get relegation. Remember that none of these reactions are 100% efficient.
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