Subcloning into lentiviral vectors - Very little to no plasmid after miniprep (Sep/20/2004 )

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

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

Hi everyone,
Please make your LB-Agar plates containing 200ug/ml Amp before transformation.Try it,good luck!

What is the copy number (or the origin) of the plasmid? Low copy plasmids can be difficult to prepare DNA from. You might consider chloramphenicol amplification of your plasmids. Make sure you are doing all of the washes in your miniprep -- nucleases can destroy your DNA during the restriction digestion if there are some left over. As a last resort, you might think about amplifying your DNA with phi29 rolling circle amplification (Templiphi).

Hi, guys,
the solution is simple, moreover, it is mentioned by manufacturer (but hidden deeply in its web site).

"This strain is endA+. A very thermostable periplasmic endonuclease will co-purify with the plasmid
DNA, possibly shearing the plasmid during restriction enzyme digestion since the endonuclease needs Mg for activity. The procedure should be terminated with a phenol/chloroform extraction or purifying the plasmid DNA on a column. If making solutions I, II, and III (alkaline Lysis, see Maniatis or Current
Protocols for Molecular Biology) for a conventional DNA prep procedure, make sure EDTA is present in
solution I."

"Different-sized colonies and some mini-preps that show LTR recombination may be observed when using the Stbl3 cells. If this occurs pick several colonies of different sizes (i.e. at least 5 small colonies, 5 medium-size colonies, and a few large ones) and analyze them with restriction enzyme digestion. Another option is to do double antibiotic selection on the LB plates with both Amp and Bsd. Remember: first recommendation is selection with 100 ug/ml Ampicillin"

So, try, folks!

I face similar problems when the colonies from streked agar plate (whcih are little old~ 2months) just wont grow if you inoculate into LB!!! and I hope u r groing STBL3 at 30C!! and in my hands compared to XL1 or DH5 the yield of pplasmid from STBL is really really less!

hi,
i have been using a plenti viral vector from past 2 months and have been facing similar kind of problem. But i have seen that things work well when u grow the plate at 37C rather than at 30C, But at the same time grow the culture at 30C, because at 37C there is a lot of possible recombinations occur due to the presence of LTRs. and do preferably use 2x amp conc