I have been attempting what should be a basic subclone procedure for several weeks now to no avail. After performing a ligation of the vector and single insert (both double-digest DraIII/SbfI), there are many colonies on the plates. I screen by colony PCR, and the vast majority of clones exhibit a band at the expected size. After experiencing issues with downstream procedures, I further screened the colonies I had performed PCR on previously with a miniprep double-digest of DraIII/SbfI to confirm expected insert and vector bands. After screening 40+ in this manner I have yet to get any results that exhibit the expected 4.1kb vector and 1.3kb insert, let alone anything that might even be explained by concatemers. What factors could lead to positive screening by PCR (reverse primer anneals only insert, forward only vector) but clearly improper constructs being present in those same colonies? Attached are an image of the PCR screen and one example of the bands obtained after miniprep double-digest of those same colonies. ladder is 1kb quanti
The most likely explanation is that your PCR bands are a result of a contamination and you did not get the right construct yet. I understand that you tried to avoid this with your primer combination. T
he choice of your enzymes is quite unusual for cloning: Both enzymes create a 3bp overhang of 3 nucleotides. Your cloning will work only in case, vector and insert have identical nucleotides in those 3 positions.
There is something not right with the digestion products. If this was a single plasmid that digested into two bands, the larger band should be brighter than the smaller one, they would be equimolar and larger bands are always brighter. So either your double digests are not working correctly, or you have multiple plasmids, or you are getting incomplete digestion, or one enzyme or site is not cutting.
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