I did colony PCR (one insert-specific primer and one backbone-specific primer) after transformation and the result showed that the construct was correct, but after that, I did a double restriction digest to further ensure accuracy and it showed there were only empty vectors, and seemed like the ligation failed. Why this kind of opposite result can happen?
Did you maintain antibiotic selection while growing the colony for the plasmid purification & double-digest? If not, the plasmid can be lost from the cells.
That's the first idea that comes to mind that explains both results.
Katie A S Burnette Yes, I maintained. I transformed the recombinant construct (pEGFP-C1 vector) into E. coli and plate bacteria on plates containing kanamycin. Then I picked colonies to do colony PCR, choosing the correct one that contained the insert to do small culture and then mini prep and double restriction digest. However, the double restriction digest result showed that there's no insert between two restriction sites while I can see the correct band after colony PCR and agarose gel electrophoresis.
How big a fragment does the digestions supposedly release? If this is a tiny fragment then you may just not be seeing it the fragment if you don't have enough DNA.
You might also confirm by PCR with the same primers whether or not your plasmid mini prep still shows the correct band by PCR.
Depending on how much plasmid DNA you used for the digest & the size of the expected bands, you might not have enough DNA to see all of the bands on an agarose gel.
What size bands do you expect? Did you do the digest of just the vector as a comparison?
It's also rare, but possible (I've seen it several times) that your insert will have a cut site for one of your restriction enzymes. If possible, check to make sure there isn't a cut site in the insert.
I think maybe your ligation failed at the first step.
The reason why your colony PCR show positive may be that your genes (both the target one and the backone) still attached on the clony.
Do you try the Blue-White Screening? If so, select the white one, and purify them twice and do the colony PCR; If positive, culture the purified colony for plasmid extraction, run the second time PCR, and restriction digestion.
Then you may get what you want.
Are you sure, that one of the restriction site has not been damaged due to cloning procedure? please check the sequence once.
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