We are using pET-32 Xa/LIC kit (Novagen 70072-3) for cloning. NovaBlue GigaSingles™ Competent Cells given with the kit are not viable, so we have used DH5α competent cells (Hi media) for transformation.
In order to check DH5α competent cells (Hi media), we have performed an experiment of test plasmid with DH5α competent cells (Hi media). Colonies of test plasmid are found on the LB plate after transformation. But after annealing with pET Xa/LIC Vector of Xa/LIC β-Gal positive control insert, no colonies are found after transformation on LB plates.
My query is: Xa/LIC β-Gal positive control Insert transformed only in NovaBlue GigaSingles™ Competent Cells not in any other competent cells (like DH5α competent cells) ?
Is there any difference between two competent cells?
I don't know about product but each company have their own company design for the particular product so in your case their might be the chance that they have design vector in such a way that it couldn't express in competent cell of high media.
I can't think of any reason why the plasmid would not be equally compatible with DH5a and Novablue. When you did the control transformation with your test plasmid, how many colonies did you get? Was it just hundreds or was the plate totally covered with colonies? That would tell you how competent your DH5a cells were. If the cells are good, then the problem likely is somewhere else.
Michael J. Benedik and Divya Tarwadi thank you both for your reply.
I got only 5-6 colonies of test plasmid when I transformed into DH5α competent cells (Hi media).
Does it mean that it can be possible for using other competent cells ( DH5α instead of NovaBlue GigaSingles™ Competent Cells), I'm not getting any transformed colonies of positive control insert?
It would seem that your DH5a competent cells are also not good (or you are having a problem with protocol). If you are using pure uncut test plasmid you should have hundreds of thousands of colonies.
Yes you should be able to use most normal competent cell strains. There is nothing special about the Novablue strain for your application.
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