pPICZA recombination in Ecoli - (Jul/07/2008 )

Hi everybody,

I am trying to amplify pPICZA plasmid in Ecoli, and I get multiple bands on the gel. It seems as if this plasmid undergoes recombination easily.
I have tried amplifying the plasmid in TOP10F', and in XL1-blue, with no success. Has somebody experienced this phenomenon? how did you solve it? I read in the forum that in 2006 Stutz has reported the same problem, does somebody know how did he solve it?

thanks,
Mirate

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E.coli strains you cite are rec- thus no recombination occurs within these cells. Now what do you mean by "multiple bands" ? What you see might be the forms that this plasmid takes... So you actually succeed in amplifying the vector, right ? and what antibiotic did you use ?

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thanks for answering. I know it is rec-, as invitrogen recommends, but in spite of that i get arround 7 bands on the gel when uncut, and at least 2 bands when linearized. i have sent the vector to sequencing, the sequenced fragment was correct, unfortunatly i realized later that the band was arround 5.5 kb instead of 3.2 kb. one explanation is that a recombination has occured in such a way. the same problem had Stutz in 2006.
the antibiotics is Zeocin 30 microgram pro milliliter in low salt LB.

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Better not use the E. coli Top10 strains, they can become resistant to zeocin.
I first used the DH5alpha strain which is also rec-, but I also got recombination. However, I thought it was due to my insert (collagen with only repeats in it). I have used the E. coli JM109 strain and it worked fantastic! The only thing is that this strain has endonucleases, so you really have to perform a phenol-chloroform extraction when you isolate the plasmid.

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Thank you very much for ur advice, it is nice to know that somebody is or was sharing the same problem, because i couldn't find any hint about it in other places, and was trying and trying for almost 6 months. Although i contacted the company (invitrogen) they did not mention such a thing as recombination problem, they even sent some of the original plasmids with many bands on the gel. I think it's shame on them, such a big company, and not telling the whole truth about the vectors, which are rather expensive.
I will certainly try your advice! But, excuse me for asking, why is it importatnt to do phenol-chloroform extraction if the strain is not end- ?

Mirate

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it's to get rid of all the protein contamination in your DNA, in endA+ there is a chance of endonuclease contamination after the prep, especially when doing hand preps, for columns preps I don't know... but do it anyway, so you won't have any more surprises

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Most column preps have an extra step with a special buffer to get rid of enonucleases (or only in special kits maybe?).

And one more tip: for most ligations you don't have to worry about ligating two plasmid fragments to each other (if your fragment is the same size as the vector fragment is after restriction), but with this vector, be aware! I was able (unwillingly) to ligate the pBluescript fragment to the pPICzA, so the bacteria were both ampicillin and zeocin resistant. Off course they also started to recombine, but still, you don't see these kind of things a lot.

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thanks again for u

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