Anybody did any AscI ligation? - (Mar/22/2010 )

have any one done any AscI ligation?
My vector is 13kb(only have AscI and PacI sites),my products is about 4kb.
I have done the ligation several times with no success.
I suspect it becauses the AscI has a too long over-hang,so what do you think?

I have used AscI in the past and had no problems. Have you tried doing a longer digestion (overnight?) to completely cut the vector and insert? I have found that a 1:7 molar (not mass) ratio of plasmid to insert tends to work for most reactions. Do the transformation and colony screen by PCR for the insert. This way you can screen 24+ colonies and run them on gel at the same time. If done correctly you can have the results of the screen before lunch.

Stuck with Ligation on Mar 25 2010, 03:27 AM said:

here you can easily calculate the molar ratios for your ligation

http://www.promega.com/biomath/

it tells you the 1:1 ratio so you just have to multiply by e.g. 7 for your final amount of DNA

I use AscI and PacI all the time. Works great. Cut it longer and then use them to singly cut a vector to make sure they still work. If an enzyme is bad, then it obviously won't clone.

tea-test on Apr 6 2010, 08:42 PM said:

here you can easily calculate the molar ratios for your ligation

http://www.promega.com/biomath/

it tells you the 1:1 ratio so you just have to multiply by e.g. 7 for your final amount of DNA