I've tried the digest two times so far, using the same SmaI (Promega, and nowhere near its expiry date), buffer, and DNA sample [which was recently purified and is in EB buffer (i.e. 10mM Tris, pH 8.5) with 1mM EDTA]. The first time, I used 6ul DNA with 2ul each of enzyme and buffer in a 20ul reaction mix and incubated at 25 degrees (SmaI's optimal temp) overnight, before heat-inactivating, treating with TSAP, and transforming 0.125ul of the mixture into E. coli. The second time, I decreased the amount of DNA in the digest to 2ul, incubated the digest for 4hrs, and used 0.25ul for transformation (didn't bother with the TSAP). I got lots (>50) of colonies both times, although somewhat fewer the second time due to the smaller amount of DNA, and when I do the actual ligation and try the transformation with that, I don't get any more colonies compared to using the equivalent amount of 'digested' vector.
Any help or advice would be much appreciated.
Edited by Orangistae, 23 April 2010 - 09:44 PM.