Ligation

[peter]

On Jun 28, 11:29 am, "Deitiker, Philip R" <pdei... from bcm.tmc.edu> wrote:
> I have repeated the ligation.
>
> 1. Positive control. Plasmid 1.6 ul/17 ul of cells.
> No ligation. No gel purification.
>
> 2. Positive control. BamH1 cut Plasmid
> no Insert, gel purified.
>
> 3. Negative control. BamH1/EcoR1 purified by
> gel and ligated with no insert.
>
> 4. Test. BamH1/EcoR1 purified with added insert
> at 3:1 ratio.
>
> The vector DNA was visible on the gel without transillumination
> and was cut out with visible light. The remainder was examined
> by Transillumination, some loss of DNA most DNA however was
> in the band.
>
> All transformations at 1.8 ul/17 ul of cells, all plates
> are.
>
> Result.
>
> 1. ~4000 CFU per plate
> 2. ~250 CFU per plate
> 3. 13 CFU
> 4. 11, 13, 19 CFU per plate.
>
> 3 and 4 are not statistically different.
>
> Conclusion.
> Problem is not primarily in  cells, either may have ligation reaction
> problems, gel cleanup problems, DNA solubility problems.
> or Insert not digested properly.
>
> I think it is worthwhile to order primers and reamplify
> products.
>
> -----Original Message-----
> From: methods-boun... from oat.bio.indiana.edu [mailto:methods-boun... from oat.bio.indiana.edu] On Behalf Of Deitiker, Philip R
> Sent: Tuesday, June 26, 2007 6:26 PM
> To: Meth... from magpie.bio.indiana.edu
> Subject: RE: Ligation
>
> "
> 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?
>
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Well, until you tell us how many nanograms of vector you are using no
one will be able to tell what is going on.
Again, looks that you don't have good competent cells, if you can see
DNA in visible light then you are using way too much of it. Good
ligations are with 1-10 ng vector, and good competent cells will give
you 1M clones per ng closed vector.
P