Problems with pBabe - unable to cut out the insert...

Tim Fitzmaurice tjf11 at cus.cam.ac.uk
Thu Aug 24 03:53:18 EST 2000


On Fri, 18 Aug 2000, Ilya Shamovsky wrote:

> is ok as evdent from restriction analysis and works fine in transfection
> experiments. However, when we made this plasmid in E.coli, it did not
> work in transfection.

Any chance of a few more details on what you mean by it did not work in
transfection. when I used this plasmid I had a lot of trouble with
handling it. Most of which we solved by not using the standard shortcuts
in these protocols.

When using pbabe, with an insert of 2.5kb, I simply had enormous problems
obtaining enough DNA from preps of any kind, even after the plasmid alone
was looked at to check it was OK, andit was.

> What is more strange is that it is no more cleaved
> by appropriate restrictases. We tried a few strains (XL1, XL2, DH5
> alpha) to no avail.

I used TOP10 cells in the end and they worked fine, I had trouble with
XL1s. I can;t help with the loss of sites, beyond the suggestion that its
something in the kits used to prepare the sample.....have you tried a big
phenol/choloroform extract and alcohol reprecipitation to remove any trace
of salts and proteins so on..
 
 At the same time, the same vector with different
> insert seems to work just fine. How come bacteria can not tolerate this
> particular DNA?

Are you getting bacteria growing, but no DNA coming back, or just no
bacterial growth in overnight cultures.

> Or is this a problem with plasmid isolation? (unlikely
> since we tried a few mini- and midi-prep kits and again, it works fine
> with another insert).

Very possibly....I had big problems with Qiagen maxipreps when I tried to
do this, the Qiagen rep and tech people were a great deal of help and we
ran through a series of things and new free kits to try and get it to
work, and in the end concluded that it was simply something weird about my
vector and insert combination, and nothing to do with the columns, the
vector alone worked fine, the insert in puC19 was fine; vector and insert
together failure.

I went back to the absolute core Maniatis protocols. Chloramphenicol in
the bacterial culutre to improve copy number of plasmid, maxiprep the
'old' way and double caesium chloride gradient to get it back....7ug/ulin
a sensible volume in the end as opposed to 8ug total off the column (yes a
maxiprep), both from 500ml of bacterial culture.......and I used LB broth
exclusively rather than the TY I'd been using up to that point, TY seemed
not to get any decent copy number, agai i have no idea why.

Doing it this way solved all my problems, and many of them were similar to
yours. 

Tim
When playing rugby, its not the winning that counts, but the taking apart
ICQ: 5178568






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