Expert: Dana Krempels, Ph.D. - 1/5/2008

Question
Hi, I am doing some research on recombinant DNA and I'd like to know why when i use EcoR1 restriction enzyme for 30 mins instead of 60 i get 3 bands on gel electrophoresis instead of just one, thanks.

Answer
Dear Emily,

This is the answer I got from my electrophoresis expert pal, Dr. Yunqiu Wang of the University of Miami Biology Department:

"To answer this question, we need to know what this student wanted to digest with EcorI.  Does the target have one restriction site or two restriction sites, meaning whether the target DNA molecule has one region with a sequence of
"GAATTC" or two regions of "GAATTC".  

"If the target only has one restriction site for EcorI, then he should always see one band whether the DNA is digested
for 30 minutes or 60 minutes, assuming the digestion is carried out at 37 degrees Celsius.  But, if the target DNA molecule has two restriction sites, it is possible that he only saw one band after digestion for only 30 minutes,
due to incomplete digestion, and three bands started to appear after the digestion time was extended to 60 minutes.  Why did the student see three bands at 30 minutes digestion instead of one band when digested for 60 minutes?  I need to
see the actual gel picture to understand why.  Did he use the same digestion buffer for the digestion at two different time periods?

It is very strange!"

If you can clarify any of the above, we might be able to tell you more.  Hope this helps get you started.

Dana