Questions about DNA recovery and cloning

Steven Goldberg goldberg at bms.com
Thu Feb 27 11:21:11 EST 1997


In article <Pine.GSO.3.95L.970226230055.22311A-100000 at uststf1>, Wilson
<netson at uxmail.ust.hk> wrote:

>         Hello everybody, I have questions to ask for your help.
> I have used GeneClean to recover DNA from agarose.  After eluting the DNA
> in TE, I discover quite serious DNA degradation from gel electrophoresis.
> I suspect the sources of contamination: TE or GlassMilk or both.  In order
> to eliminate the problem, I autoclave the TE and the question is, is it
> possible to autoclave the GlassMilk without affect the DNA-binding
> activity?  Is there other possible sources to get the contaminant?
>         The second question is what density of the E.coli (OD600) suitable
> to make competent cells?  Is it necessary that the higher OD (e.g. 6.0),
> the lower transformation efficiency as a result?


Are you sure your degradation is due to a nuclease?  If you handle DNA
bound to GlassMilk too roughly (e.g., by vortexing or pipetting too
vigourously), you can shear your sample.  This is especially true for
larger fragments.  Because of this problem and difficulties using
GeneCleaned DNA in ligations, I recently switched to the QIAquick Gel
Extraction Kit.  The DNA is very clean, shows no signs of degradation or
shearing, and performs very well in labeling and ligation reactions.

Competent E. coli is best made from cells just into logarithmic growth
(around OD600 0.5-0.8)

Good luck.

Steve



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