Anybody did any AscI ligation?
Posted 22 March 2010 - 06:05 AM
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?
Posted 24 March 2010 - 11:27 AM
Posted 05 April 2010 - 05:41 AM
Thanks，how do you convert the mass to molar ratio ?
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.
by the way,my vector is 13kb and insert 4kb..
Posted 06 April 2010 - 06:18 AM