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why a low copy number? - shRNA vector (Feb/06/2008 )

Hello,

So the commercially available vector that I have subcloned my shRNA into is a very low copy number vector. Why, in this age where we can pick which ever ori we wish, is it important that this vector be low copy number? I know that it is possible to increase the copy number by spiking my broth with chloramphenicol. Is that an advisable thing to do? I ask because on the one hand I need sufficient plasmid to make virus, but on the other hand if there is a reason that the vector is a low copy number vector then maybe adding the chloramphicol will all the R&D that went into making the vector.

Also, I'm new to shRNA, could someone explain to me what all the buzz is about with chosing a rec- bacteria to grow up the plasmid in? Why is recombination such an issue to shRNA plasmids? And if recombination can occur should I get my plasmid sequence after doing a maxiprepp (or from my initial miniprep )?? If this plasmid says on its spec sheet that I can grow it up in regular DH5alpha, then do I still need to be worried about recombination??

Thank you in advance!!

-stemcell69-

Hi,

for just cloning shrna into a plasmid the rec- is not so important, same with low copy number ori. The suggestions are, correct me if i'm wrong, mainly for cloning shrna into retro- or lentiviral vectors because the recombination frequency between the long terminal repeat (LTRs) is quite high. Low copy number also seems to help against recombination events. Introducting a complementary sequence like shrna between the virus LTRs pushes up the recombination frequency even more it seems. So for cloning shrna into a non viral plasmid is a lot easier than cloning into a viral one. I never did chloramphenicol amplification myself, i just increase the culture size (500 ml instead of 300 ml for maxi prep). I always do a control digest of new maxi preps to see if the plasmid is still ok. The first maxiprep i always sequence it. This is very important for shrna sequences especially if you get the sequence synthesiszed as ss DNA oligos or generate them by PCR.

Stardust

-stardust-

Many viral vectors have terminal repeats which can undergo recombination and actually modify the vector. i have seen cases where the TR's are missing on one side and you donot get virus production. The same for shRNA. Its better to have them in rec- cells. We verify plasmids after maxipreps.
I would be worried if this vector has terminal repeats even if the spec sheet says it can grow in DH-5alpha.
To overcome the problem of low copy, we usually do a 10ml day culture and use it to inoculate a 250 ml or 500ml culture depending on how much you need. We get about 1-1.5 mgs of DNA.

-scolix-

Thank you ! I appreciate your comments.

-stemcell69-