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Subcloning into lentiviral vectors


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#1 egegik

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Posted 20 September 2004 - 02:04 PM

Hi - I am trying to subclone into a lentiviral construct into which I have succesfully subcloned into before. For some reason I have lost my mojo however:

Transformation of ligation gives 5x more colonies (Stbl3, HB101 derivative, needed to tolerate the LTRs in the lentiviral genome) from vector + insert ligation. When I innoculate of these colonies into LB, the bugs grow (very very slowly as is usual with Stbl3 cells) but upon Qiagen minipreping I get either very very little plasmid or no plasmid (after digesting and running on gel).

The admission: one of the times when I got no DNA I had innoculated from a plate that was ~2 weeks old (stored at 4C though) but this weekend I restruck a colony that looked positive before (weeks ago) and innculated from a single fresh colony and got nothing again on my miniprep.

Are plasmids contained in HB101 derivatives sensitive to sitting on plates for a while?

Desperate,

Jeff

#2 Ulli

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Posted 20 October 2004 - 10:49 AM

Hello Jeff,

I had the same problem with disappearing plasmids after Mini Prep and restriction digest. I assume you had also a undigested control on your gel? I tried these once and I had a positive signal from my undigested vector but not after digestion. And, for everyone who might argue, I could have forgotten to add plasmid to my digestion: Have you ever forgotten to add plasmid to 10 tubes? Anyway. I had no plasmid anymore. My technical assistant gave me the advise to run the plasmid over the gel, clean up and try a digestion again. Usually this should hepl. I don`t have a result yet, but I`ll try tomorrow. If this is the case, Stbl3 cells must contain an enzyme with which they can digest DNA and it might not be sufficient to clean up your DNA with a usual Mini-Prep.
Do you have any results yet?
Looking forward to get some information.
Thanks for every advice.

Ulli




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