Cloning with Tth 111 I

LouPass loupass at aol.com
Sat Mar 8 19:42:34 EST 1997


I have used this enzyme several times.  Normally we run digestions at 65C
with a little oil overlay (similar to PCR).  I would suggest that you add
excess enzyme during the digestion and let it go for at least 3 hours (for
approx 1 ug of DNA).  For ligations, I usually carry them out with about a
2:1 ratio of insert to vector and use excess ligase at 16C for at least 24
hours.  Alternatively, you can try to blunt the ends with Klenow or T4 DNA
Polymerase and do your ligations with that.  I have found that even with
vast excess of enzyme digestion was never complete so make sure you have
good controls and size standards so you can pull out the band you want. 
Good luck.

Lou Passador
Univ. of Rochester.



More information about the Methods mailing list