Blunting 3' ends with T4 polymerase - (Oct/11/2001 )

What is the best protocol for blunting a 3' overhang with T4 DNA polymerase. Please reply with methods that ROUTINELY work for you. The sequence I am degrading is 5GAGCT-C3 (Sac1 site) in preparation for a blunt end ligation. So that the 5G is all that will be left. Should I omit all nucleotides except G and C or is this not necessary.

<p>Brian EsauNCSU, Raleigh

Just use all the nucleotides, 12 degrees C for 10 mins then inactivate the enzyme at 75 deg C for 15 minutes, it worked for me!