Help with Cloning

[Austin P. So (Hae Jin)]

Prema wrote:

> 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
> "normalizing"
> restriction enzyme activities to "pmol sites per hour", and then
> figure  out how much would be needed at a minimum for whatever I would
> digest"
> 
> With reference to the above suggestion could you tell me how to
> normalize.

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 
sufficient, etc...

Cheers

Austin