minipreps from colonies

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Wed Aug 11 08:57:15 EST 1993


In article <CBKF3n.75q at ncifcrf.gov>, pnh at fcsparc6.ncifcrf.gov (Paul N Hengen) writes:
> In article <1993Aug10.093228.2704 at gnv.ifas.ufl.edu>
>  afc at gnv.ifas.ufl.edu writes:
> 
>> We have been working with libraries in Promega's phagemid lambdaGEM-4
>> vector.  After popping out the insert + plasmid (pGEM-1) we transform
>> into DH5alpha, plate on ampicillin, and pick a single colony.  We then
>> restreak this and have our subclone.  This works great.
>>
>> However, if we try to scrape some cells off of the restreaked plate to 
>> do a miniprep, we get no DNA.  About ten clones of different genes from
>> different species, using either alkaline lysis or boiling, have failed.
>> If we grow the cells in broth, no problem, lots of DNA.  I have been doing
>> minipreps off of plates for ten years and never had any problems before 
>> this.  
>>
>> This is not a major problem, but it is one for which I cannot come up with
>> a logical explanation.  I presume that it has something to do with the
>> vector, the host, or some interaction between the two.  Anybody know the
>> answer?
>>
>> Andrew Cockburn
> 
> If the plates with DH5alpha colonies are old or stored at 4 C, the cells
> produce a fair amount exopolysaccharides which might interfere with your lysis
> procedure.  Maybe you could try resuspending the colonies in 1 M NaCl as per
> the mini-prep method I've posted previously. Also, I find that the hot alkaline
> method helps the lysis of this strain. I think you would have no problem with
> isolating the plasmids from colonies this way.
> 
> Paul N. Hengen
> National Cancer Institute
> Frederick Cancer Research and Development Center
> Frederick, Maryland 21702-1201 USA

We used only freshly streaked colonies, e.g. less than one day old.  Poor
cell lysis is something that I hadn't considered, however we did see RNA
in some of the minipreps.

Andrew Cockburn



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