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recombinant bacmid has double bands
Posted by: SW (IP Hidden, New member, 3)
Date: August 29, 2005 09:46AM
hi
i am trying to produce proteins using the baculovirus protein expression. my gene of interest was succesfully inserted into the pFastBac1 vector and confirmed by sequencing. the pFastBac1+gene construct was then transformed into DH10Bac E.coli for producing recombinant bacmid. White colonies was recovered and screened by by PCR M13-40, as recommended by protocol. The funny thing is, following miniprep, the recombinant bacmid DNA gave double bands, which are quite close to each other. I picked out ALL white colonies from the DH10 transformation plates and they all gave double bands. My question is, does the presence of this other band would interfere in the subsequent transfection step? If yes, how do I get rid of this other band? I have re-transformed the pFastBac1+gene construct into DH10 E.coli and still all the white colonies gave double bads. In addition, I did not get double bands for my other constructs (different gene of interest) in DH10..please advice. thanks.
Re: recombinant bacmid has double bands
Posted by: backu (IP Hidden, Unregistered user, )
Date: August 29, 2005 12:01PM
Could this be just different migration from your bacmid DNA, possibly due to the conformational differncy? Does the sequence of the gene give difficult secondary structure? Just a thought.
Re: recombinant bacmid has double bands
Posted by: vishal (IP Hidden, Unregistered user, )
Date: August 30, 2005 06:09PM
You saw double bands in PCR amplified product ? Or did you analyse your bacmid minipreps as it is on agarose gel ? I am facing a different problem here. I could'nt see my protien expression even after 2nd round of amplification. Not to mention that i have checked my bacmid for gene of interest through pcr using M13 primer pair. Sorry for asking question on question. Vishal
Re: recombinant bacmid has double bands
Posted by: SW (IP Hidden, New member, 3)
Date: August 31, 2005 08:46AM
my PCR product using the M13 (-40) primers gave one band and correct sequence. The double bands were in the bacmid miniprep. I actually wrote to invitrogen, and its staff said, in order for the transfection to occure correctly, the bacmid miniprep product must be a "single" band. Earlier, I also went ahead for transfection into Sf9 cells following confirmed PCR (of this double band bacmid), and did not get any protein production. That is why I began to wonder if this "extra" band interfere with my transfection. The invitrogen staff said, "yes"; a question of picking a mix of recombinant and non-recombinant colony. And that is why I reversed and re-transformed the pfastbac1+ gene construct into DH10Bac...still double bands in ALL white colonies, and if these white colonies were re-streaked onto another plates, still double bands bacmid.... What could be the problem?
Re: recombinant bacmid has double bands
Posted by: jonmoulton (IP Hidden, Junior member, 15)
Date: August 31, 2005 12:07PM
Hi SW,
I took a look at the Bac-to-bac manual from Invitrogen and saw that the bacmid is circular DNA. If circular DNA does not have a nick in one strand, you can have a population of supercoiled plasmid and a population of relaxed (nicked) plasmid that migrate at different rates in agarose. Try digesting a bit of your miniprep with a restriction enzyme that has a single lytic site on your bacmid and running another gel. If the linearized plasmid runs as a single band, then it is a reasonable hypothesis that your circularized plasmid is running as a supercoiled band and a relaxed band. - Jon
Re: recombinant bacmid has double bands
Posted by: umea (IP Hidden, New member, 1)
Date: September 30, 2005 02:39AM
Hi.
We are also struggling with the bac-to-bac system, and I must say that I am getting pissed. The reason that you get the double bands is that there is no active selection against everything else than the bacmid. What you see is plasmid DNA. Either Helper plasmid, your pFastBac1 plasmid, or both. They even say that one should include tetracyclin in the overnight culture, which will lead to a continous selection to keep the helper plasmid. And even if there is no selection for ampicillin, it is not certain that your pFastBac1 plasmid is lost. I have asked them about this today. Will see what they say. There should be some kind of active selection against everything but the bacmid. Best wishes, Stefan
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