Ligation

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Wed Jun 27 12:57:54 EST 2007


On Jun 26, 7:25 pm, "Deitiker, Philip R" <pdei... from bcm.tmc.edu> wrote:
> "
> Seems to me that you also look way to long under the UV.
> "
>
> I have a helper, we cut bands from one side to the other side
> of the gel, I do all the 'vertical' cuts based on lane separators
> and make a single quick top cut under LW-UV, followed by one by one bottom cuts, with side exposure. As soon as I make the bottom cut she moves the light away and we remove the band and move to the next lane. The process is very fast, but that is why we messed up a couple control lanes.
>
> Those samples have been discarded, I have now made up several
> times more RE digested vector without CIPing and some more BamH1 vector. For the exact reason you mention, they may be fine for diagnostics but I would not subclone into them.
>
> "
> If you use DH5a, then you have the control plasmid (pUC) that comes with them.
> "
>
> pUC-19 actually, I threw it away Sunday when I cleanedup the freezer.
> I have lots of plasmid (see below), will run the control next.
>
> "
> If it is good then having 150 colonies from uncut vector  is way too low.
> "
>
> Right. As much as I suspected. I have transformed cells recently
> (1 ul) for the purposes which were not UV exposed and I am not
> getting 1000s of colonies, more like 20 colonies per microliter of cells of pure vector. The cells themselves are viable, on nonAmp
> plates I get 10,000s of colonies per 0.1 ul of cells. Though I have not checked them recently so . . . .
>
> If it does not transform efficiently I will buy new cells, these are the last of a batch that is about 2 to 3 years old -80'C, and I am on the last aliquoting of the stock. Apparently they were moved to another
> freezer and after I made a fuss about it they were moved back so . . . .
>
> Thanks for the help, at least now I know what the pUC-19 was for in
> the box with DH5alpha :^).
>
> Somewhat new problem.
> I am using Promega's Midiprep kit, it appears even after attempts
> to dry off the ethanol the resulting DNA has EtOH, or at least, does not freeze and has the trace smell of Ethanol. Last time I did an EtOH
> precipitation on the midiprep DNA, problem is that much of the precipitated material did not resolubilize, possibly because I incubated it too long at 65'C I am weary of doing this again so we were really careful to dry the membrane, but I still recovered a great amount of DNA. Has anyone used the
> Promega Midiprep kit and had similar problems?
>
> Also, apparently the tube cultures of bacteria seem to work fine, but a similar volume of flask culture seems to overwhelm the kit. Too much
> DNA? Is it possible to clog the DNA binding membrane with too much DNA?

Well, I am not surprised that your sample does not freeze (maybe if
you shake it it will at once) . In any case if you have residual
ethanol it won't prevent freezing at this concentrations. The quick
way to tell if ethanol is present is to load on gel - if it floats
than it is present. As for having too much bacteria in midiprep, as
soon as you have complete lysis  of the cells then it should work just
fine.
BTW make (buy) new competent cells and always keep the control
plasmid :-) , also read the insert as to how to perform control
transformation and the following estimations.
Best,
Peter



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