problems with restriction digestion

[Michael Sullivan]

Another thing to keep in mind is how you incubate the digestion  
reaction. Lots of people incubate digestioin reactions in a  
waterbath. Be very careful doing this, especially with enzymes that  
have star activity. In a waterbath, what often happens is that the  
bottom of the tube is warm, but the top of the tube is cool. Water  
evaporates from the reaction and condenses on the top, increasing the  
concentration of enzyme, salt and glycerol in the digest. For small  
volume digests this effect can be significant, and I've certainly  
seen BamHI digestions exhibit star activity when incubated in a  
waterbath. I prefer to carry out my digest incubations in a warm air  
incubator: initial heat transfer to the reaction is slower, but there  
is no problem with evaporation/condensation since warm air surrounds  
the entire tube.

Mike


On Feb 8, 2008, at 12:09 AM, chemophoto from gmail.com wrote:

>
>
>  I used a 20ul digest, 1ul restriction enzyme, 2ul buffer and 1ul of
> plasmid DNA. I do not know the concentration of my plasmid DNA (didn't
> nanodrop it yet). I used 1ul plasmid DNA based on the agarose gel
> result of the midi prep.  I digested it for 2hrs at 37 and did an EtOH
> pption and did the second digest.
>
> I am wondering why I am getting extra bands. Am I using too much
> enzyme? But I have used this amount of enzyme in digestions of 20ul
> before and it worked.  Is there something else that I am doing
> wrong??
> I thought the reason why I got the extra bands before was as I was
> using an alkaline lysis prep..which might have contaminants (RNA
> etc).
>
>

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)