We had to put our pJet1.2 plasmid in a XL1-Blue Competent Cells. The result were very small colonies on a LB agar medium with ampicilin and IPTG. bands with a very low concentration were visible after gel electrophoresis. Is there a different bacteria that we can use that can produce more plasmids so we can see it with gel electrophoresis?
Hi Niels!
In my laboratory we tend to use a competent strain of E Coli called DH5 alpha, you can buy it easily in ThermoFisher in the link I attatch as follows: https://www.thermofisher.com/order/catalog/product/18263012
The transformation efficiency is pretty good, and the protocol is easy (with CaCl2 and freezing thermal shock).
You have 3 different ranges of quality regarding this DH5 alpha strains.
I hope this information helps you!
Best regards,
Francesc
Are you wanting to transform the native pJET1.2 to make more vector, or is this a clone with an insert in it?
If you are wanting to propagate a clone, then nearly any E. coli strain should be fine, DH5alpha is pretty typical but XL-1blue will also be fine. I don't think you will see much difference between the strains.
However if you are trying to propagate the native pJET1.2 without an insert, then you need a very special strain because pJET carries a lethal insert. So you need a strain expressing the correct methylate to let the plasmid survive.
Do you use IPTG because you are screening for blue/white colonies? Have you tried without IPTG, this means not using the blue/white selection? Colonies could grow bigger without, because they do not have to produce the alpha-peptide fused to the peptide encoded on your DNAinsert.
Best wishes, J
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