Anybody did any AscI ligation? (View forum version)



AllenChiu

Posted 22 March 2010 - 06:05 AM

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?

Stuck with Ligation

Posted 24 March 2010 - 11:27 AM

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.

AllenChiu

Posted 05 April 2010 - 05:41 AM

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.

Thanks,how do you convert the mass to molar ratio ?
by the way,my vector is 13kb and insert 4kb..

tea-test

Posted 06 April 2010 - 04:42 AM

here you can easily calculate the molar ratios for your ligation :rolleyes:

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

Stuck with Ligation

Posted 06 April 2010 - 06:18 AM

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.

AllenChiu

Posted 06 April 2010 - 07:25 AM

here you can easily calculate the molar ratios for your ligation :rolleyes:

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

Thanks a lot~