Ligation

[IanMc]

"peter" <peter.ianakiev from gmail.com> wrote in message 
news:1182970154.634835.88750 from q69g2000hsb.googlegroups.com...
> On Jun 27, 1:57 pm, peter <peter.ianak... from gmail.com> wrote:
>> 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
>
> Also, please stop referring colonies per microliter , if you would
> like to make sense use colonies per microgram (nono-, fempto- etc.)
> plasmid.
> P
>
I guess that would be nano- and femto-  then  :-)

IM