Hello
I recently transformed bacteria with my vector and insert ligated construct. In order to check the presence of both the vector and insert, I mini prepped 20 bacterial colonies and did restriction digest with NotI. But, the gel shows an extra band in between the vector and the insert. My insert is 1.1 kb and the vector is around 5kb. I have attached a picture of the gel below. I used Instant sticky end ligase from NEB with 1:4 vector:insert ratio. Could this be a contamination? Is the insert ligating with another insert?
My vector has single NotI restriction site. I should only be getting two bands here. Your suggestions would be really helpful!
- Thank You!
What is the size ladder please?
There is no uncut control sample so possibly the largest band is genomic dna unable to separate properly due to its great size
It looks to me as if the plasmid is not digesting completely, but it could also be genomic DNA that is not well digested as suggested by Paul Rutland . Try selecting one or two of the mini-reps and run controls, uncut, cut with NotI and cut with another enzyme that digests the plasmid one time.
It looks like most of your DNA is not being cut by the restriction enzyme. You seem to have a lot of DNA in those lanes, based on fluorescence intensity relative to the ladder. Try using much less DNA, maybe 5-fold less, and see if that helps.
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