Help with Cloning
Austin P. So (Hae Jin)
nobody at nowhere.com
Thu Sep 30 14:10:44 EST 2004
> 2. how much are you digesting?
> I did a miniprep and digested around 5 ul of DNA in a final volume of
> 20ul. I did not spec the DNA for concentration. Also the miniprep did
> not include RNase treatement. I use 1ul enzyme and usually digest for
> 2 hrs.
> 3. how much of your digest are you loading onto your agarose gel?
> I load all the 20 ul on the gel to cut out the bands.
Okay...I think it would be prudent to quantitate your plasmid. That will
be able to tell you many things. There are many ways to do this, so I'll
leave that up to you.
Generally you shouldn't load more than 1 microgram of material on an
agarose gel per lane (I usually add no more than 500 ng if possible).
If you still have a problem, then something else is going on which is
not obvious...everything else you have covered well...
> 6. "P.S. what I tried to do early on is to get in the habit of
> restriction enzyme activities to "pmol sites per hour", and then
> figure out how much would be needed at a minimum for whatever I would
> With reference to the above suggestion could you tell me how to
Enzyme activity units are expressed as "1 U cuts x microgram of template
DNA per hour at a final volume of x microlitres".
So you just find the number of sites in the template, calculate number
of moles template, and voila! you have an estimate of its activity in
"pmol sites per hour" which you can use to compare across all
manufacturers...it also will give you an indication as to whether or not
you are adding to little, or too much, or if the incubation time is
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