I am trying to transform a pre-made plasmid in the E.coli BL21. The plasmid has my insert (potential promoter sequence) upstream of the RFP protein. Usually, all my positive clones are pink in colour. After the transformation of the particular plasmid in E.coli BL21, I found only white colonies and PCR showed no band for my insert. I wonder why my insert is deleted from the plasmid? And, what strategies should I follow to keep my insert before the RFP gene?
The fact that you can get pink colonies from other constructs means that RFP is not toxic, per se.
Have you tried sequencing or a restriction digest to see if your insert is present? PCR directly from a plasmid does not always work.
I'd try the transformation again with a full set of controls to see if you can narrow down the possibilities. The most likely is just low transformation efficiency.
Also, I use the "verify, don't trust" mindset when getting reagents from someone else. Check the plasmid for the presence of your insert BEFORE you try the transformation.
Katie A Burnette Thanks for the answer. I made the construct in E.coli Dh5a. Picked the positive clones (pink), extracted plasmid, PCR confirmed and then sequenced it. After that, the plasmid was transformed into E.coli Bl21. There, I found no pink colonies and colony PCR shows no positive band for my insert. I am wondering what would be the reason behind losing insert in BL21?
Saadlee Shehreen
Something must be missing from your description. If the plasmid that you transformed was toxic in BL21 , then you would expect to simply get no transformants, because they would all be dead--or at least grow very poorly. If somehow, what you transformed was actually a mixture that also included some plasmid vector without insert, then you might get the result that you describe. When you did the BL21 transformation, did you get as many transformants as you would have gotten with the vector plasmid alone? We don't know the details of how the insert was cloned into the vector, but the possibility of frequent clean excision of the insert would seem very unlikely.
Saadlee Shehreen
One possibility that comes to mind is that you might have picked a dimer plasmid that consists of one copy of the vector plus insert and another copy of the vector alone. Such a dimer could resolve rapidly into a monomer of vector only Did you pick more than one of the original clones to analyze? Did you run uncut plasmid DNA from several clone candidates on a gel and compare it to vector plasmid? If you just cloned a promoter, then the plus insert plasmid should be only slightly larger. If you compare several clone candidates, you might see that some of them are much larger and these might be the type of dimer mentioned.
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