E-coli strain and plasmid incompatibility...? - is there something like that? (Aug/29/2006 )
i have electroporated my plasmid into BL21 strain of E-coli. Tried to do miniprep and getting very strange results. BL21 has recombinase i guess so I shouldnt have used it right? or is there something like this vector type is not good for this E-coli stain? in general i mean. thanx a lot.
Maybe it's something in your plasmid. I had it once when coli recognized some kind of promotor-region in my insert (wasn't expecting anything like that and it's sure wasn't ment this way) and started to translate against the direction of the ORI, and that gave problems.
Does the BL21 strain also has a F'? That will help sometimes.
in general we use DH5a or Xl1blue strains caus BL21 can recombine.
But in general BL21 are better for protein expression in our lab.
what do you mean by 'strange results'? could you please post a pic of your miniprep gels?
Minipreps from BL21 are problematic, because the strain is endA+, and expresses endonucleases which degrade DNA during/after the preparation. High quaility plasmid should be prepared from endA- strains, such as DH5a or Top10.
thanx a lot everyone!
im not sure what if F'....i know it doesnt have it but is it to prevent recombination?
amikins, i have either no yeild or very low yeild of plasmid. i think what phage has said explains my results..
phage, i am not sure but if i have the "origami" DE3 strain of BL21 it doesnt have the endA+
Origami™ host strains are K-12 derivatives that have mutations in both the thioredoxin reductase
(trxB) and glutathione reductase (gor) genes, which greatly enhance disulfide bond formation in
the E. coli cytoplasm. Origami B host strains are derived from a lacZY mutant of BL21 to enable
precise control of expression levels by adjusting the concentration of IPTG. The mutations in
trxB and gor are selectable on kanamycin and tetracycline, respectively; therefore, these strains
cannot be used with plasmids that can only be selected with kanamycin or tetracycline. These
strains also include the lon and ompT deficiencies of BL21, which increase protein stability.
please download this and read it its great
http://www.bio.indiana.edu/~ybelab/procedu...gami%20cells%22
If you read the Novagen competent cell genotypes, you will see that all the Origami strains are endA+. I should also mention that the B strains of E. coli and their derivatives are substantially less competent, by 100-1000x than the standard cloning strains such as DH5a, Top10, or Novablue (if you like Novagen). So you don't want to be trying to clone ligation results into them, unless you are feeling lucky. The Origami 2 strain, which I have not worked with, appears to be missing the deletions necessary for plasmid stability and high transformation efficiency as well, even though it is a K-12 derivative.