I have done an electrophoresis of white colonies from blue white screening. Why are there non-recombinant plasmids bands?

Dear Emilie,

The solution may be very simple: if you clone in Lambda DNA digested with PstI into PstI-digested pU19, and then digest all of the plasmid DNA samples produced from cultures inoculated with blue or white colonies and selected with ampicillin, then all of your samples will include a band at 2.7 kb corresponding to ... pUC19 ... which is the source of the ampicillin resistance and will turn the colonies blue if there is no insert of DNA at the PstI site.

Your gel images suggests that a number of your samples contain Lambda DNA inserts, but since the band intensities vary a lot, I'd be a little careful in interpreting some lanes with bands above the pUC19 band, as this may represent partially-digested plasmid DNA. Lanes 3, 6, 7, 9, 10, 13, 15 and 16 look interesting. You could look at a Lambda restriction map to see the range of PstI fragments it would produce, and map them onto the fragments you can see in your gel.

Regards, Andrew

Nikolay Klyashtornyy

If you are observing bands similar to pure pUC19 in the gel electrophoresis of white colonies from a blue-white screening, there are a few potential explanations for this observation:

Background Recombination: Blue-white screening is a widely used method to identify recombinant colonies. However, it is important to note that the screening is not 100% specific and can sometimes result in background recombination. Some white colonies might still contain non-recombinant plasmids, resulting in the presence of bands similar to pure pUC19.

Vector Self-Ligation: During the cloning process, self-ligation of the vector backbone (pUC19) can occur, resulting in circular, non-recombinant plasmids. This self-ligation can happen even in the absence of an insert DNA. The presence of bands similar to pure pUC19 in the gel could indicate the presence of such self-ligated plasmids in the white colonies.

False Positives: Occasionally, blue colonies can arise due to factors other than successful recombinant DNA insertion. For example, spontaneous mutations or recombination events in the lacZ gene of the vector can lead to the production of β-galactosidase enzyme, resulting in blue coloration. In such cases, selecting white colonies may still result in non-recombinant plasmids being present.

To further investigate and confirm the presence of non-recombinant plasmids in the white colonies, you can consider performing additional analyses such as:

Plasmid Isolation and Restriction Digestion: Isolate plasmid DNA from the white colonies and perform a restriction digestion using appropriate enzymes. Analyze the digestion products on a gel to confirm the presence or absence of the desired insert DNA.

Sequencing: If possible, perform DNA sequencing of the plasmid DNA from the white colonies to directly confirm the presence or absence of the desired insert. This will provide definitive information about the identity of the plasmids.

Control Transformations: Perform control transformations using known recombinant plasmids and empty vector as controls. This can help identify any issues with the blue-white screening method and provide further insights into the presence of non-recombinant plasmids in the white colonies.

By combining these additional analyses with your gel electrophoresis results, you can obtain a more comprehensive understanding of the presence of non-recombinant plasmids in the white colonies and further optimize your cloning experiments if necessary.

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