I'm now working on a large quantity of DNA cloning which have to repeatedly perform mini-prep and midi-prep. I was been told that some of the plasmids such as pUC57 or pLNCX can first be grow in 3 c.c. LB in polystyrene round bottom tube, half of volume will be use to extract DNA and enzyme check, and if the result was correct, 20 ul of LB bacteria mixture can then be transfer from the rest 1.5 c.c. to a new 100 c.c. LB containing Erlenmeyer flask, than midi-prep.
But only for the plasmids size that's about 5000 bps. For a larger plasmid such as pLKO that in our lab is around 9000 bps, this procedure can not be done.
That is we must do the mini-prep than re-transform the correct DNA, we can than use it to perform midi-prep.
Does someone have theory or experience about it?
I can think of no valid reason why size alone would be a factor in your ability to passage a culture as you describe. I think the same protocol would work fine for both size groups. Having said that, if you have a clone(s) that is showing a growth defect, the more times you grow out a culture you increase the likelihood of picking up mutations that alleviate the growth defect. But that is normally more an issue with expressing some proteins and not so much a size issue. It might also be there is trait with the pLKO plasmid that might be selected against, but again this is not a size issue but rather a plasmid genotype/phenotype issue.
Michael J. Benedik Thank you! base on your information I’ll try my own to test the efficiency and fidelity of DNA product between direct transfer and re-transformation.
agreed, there is no reason that larger plamids will grow any different. We routinely grow plasmids as large as 15-20 kb and innoculate 500ml cultures rom 2ml starter cultures with out any problem. Very large plasmids may give you a smaller yield in terms of micrograms, but they will still grow no matter how you innoculate your flask. Don't know where you heard this but it is tripe....
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