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Questions about DNA recovery and cloning

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed Feb 26 18:10:11 EST 1997

At 11:13 PM 2/26/97 +0800, Wilson 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.

Are you being gentle with the procedure? Other than DNase contamination, vortexing or vigourous up & down pipetting of the glass-milk, after binding the DNA, will surely result in a gel image that looks degraded (sheared)

>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

These are simply finely powdered glass particles. So, it should be possible to autoclave it.

Is there other possible sources to get the contaminant?

Not unless the gel aparatus and/or one of the components had been contaminated heavily with DNase. We simply wash our sumbmarine aparatus with the running deionized water to prepare gels for analytical purposes and have never had any problems. 

>	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?

Yeah! The OD directly relates to the point in the logarithmeic phase of the growth curve. The stage of the growth the bacteria in the culture will determine their subsequent acquisition of competence.

Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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