Hi all,
I've never worked with E.coli strains that already carry a plasmid and tried to make competent cells from them.. is it simply the usual protocol for chemically competent cells, but add the appropriate antibiotic in each step?
Your advice will be much appreciated
Cheers
Lisa
Hi
First.Under normal conditions, U can buy such E.coil with the pLysS or pBirA.
If U want to make it yourself, the first thing is to obtaining pLysS or pBirA or any plasmid and transform it into BL21 or JM109 etc.
This (two) step(s) will do require appropriate antibiotic in solid media and liquid media.
And the protocol of make that E.coil competented is the same as normal E.coil (none-resistant).
I will recommend that you buy the E.coli with the pLysS or pBirA optimizations, so that you are sure of what you are working with, as you have not worked with E.coli carrying plasmid before.
Thanks
Hello, In theory yes, add the appropriate antibiotics to keep the plasmid to make competent cells. Usually, for protein expression, the cells do not need to be super competent as the constructs have already been cloned and verified...just need to transform into a new host.
In this case, for simplicity, I would suggest any of the common TSS methods...they're quick, easy, and will work well for already made plasmids.
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