Greetings,
I have recently been considering the creation of a new competent E.coli strain for further research. I'm interested in incorporating a plasmid into this strain(~10Kb in size | replication origin is p15A ori | express 4 proteins controled via pBad system | Chloramphenicol resistent).
However, I am concerned about potential issues that might arise when I attempt to transform it with another plasmid that I need to extract(~5.2Kb in size | pUC/Origin | AmpR). I aim to extract this plasmid as purely as possible for mammalian cell transfection. I'm worried the incorporated plasmid may contaminate my plasmid extraction process.
While I could attempt to make the bacteria lose the ~10Kb plasmid by withdrawing the antibiotics, I believe this will be a time-consuming process. I've read in some articles that it might take approximately 30 generations for a bacterium to lose a plasmid.
There are two questions I would appreciate some input on:
1. When using a competent E.coli strains such as Rosetta (which includes pRARE) or LEMO21 (which includes pLEMO) to amplify plasmids, is the potential contamination from those innate plasmids a consideration?
2. I am wondering if it might be possible to use DNA size selection methods such as magnetic beads to extract only the smaller plasmid, which is approximately 5Kb in size.
Easiest approach is just run your extracted plasmid on an agarose gel and perform a gel extraction of the desired band.
Also why do you need to transfect with both plasmids? Maybe consider trying to integrate your one plasmid into the genome using a transposon if you do not want it contaminating further manipulations.
Thanks alot Brinzer! I shall consider purifiy it.
Indeed, making it on the same plasmid could be a great idea. However I need to transform and extract it multiple times with some PCR mediated mutagesis, having too much genes might cause addition problems.
Maybe I will consider try to integrate into bacteria genome if needed.
Thanks again!
If you are gel extracting your band then usually a contaminating plasmid is not an issue. But yes the DNA is likely to always be present. If this is an issue, another solution would be to do a mini prep and then retransform some clean cells and select only for your Amp plasmid. Most of the transformants will only pick up the one plasmid (but you should test and confirm before proceeding).
But to specifically answer your question, yes you are going to get some cross contamination with Rosetta pRARE and similar. However these are often low copy number plasmids so that amount of cross contamination is not likely to be high enough to be a problem.
I don't think size separation by most methods is going to work in this size range (5 vs 10kb), other than by gel.
I think it is better to confirm the presence of the desired plasmid by running the extraction product on agarose gel after extraction. Then you can purify your plasmid in different ways, such as gel purification
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