SmaI/XmaI digests

[George Theodoris]

Dr. Pamela Norton (P_Norton at CALVIN.JCI.TJU.EDU) wrote:
:         I am literally tearing my hair out over this one. Does anyone know
: whether Sma tends to nibble 5' or 3' ends? 

Answer is: Yes. From what I understand it can be lot dependent. Where do 
you get your Sma from? I was doing a similar experiment a couple of 
months ago and it worked fine for me. Sequencing revealed no bases were 
chewed back. However before the experiment I was made aware that SmaI is 
notorious for chewing back bases, so I was very careful about the amount of 
Sma I added and the source of the Sma I was using. We get our Sma from New 
England Biolabs. Their Sma is purified from recombinant E. coli and is 
less likely to have DNAses than lots purified from Serratia. Check and 
see if the Sma you are using is purified from recombinant E. coli or 
Serratia. Even so, one of the NEB people told me that all lots of SmaI 
contain traces of DNAses that can chew back bases. You have to add the 
minimal amount of SmaI needed to cut your DNA. Adding more enzyme than 
you need will increase the concentration of DNAses and make chewing back 
more likely.

: In other words, would it help if
: we try to refill any nibbled ends before ligation? 

This might help. But I would think you should be able to get a batch of 
Sma that works. Good luck!		-George