restriction to ligation - (Jun/25/2006 )
OKAY...this is fun...I am a first time user...can you tell?! Well...my second question is ...I am wanting to test my enzymes and vector so I am doing some restriction digests which I would like to follow with a test ligation....I guess it needs to be cleaned up some....would anyone even consider going from one straight into the other???
What I was thinking was a quick P:C:I and EtoH recipe...
that should work
yes, you do need to remove the restriction enzymes and digestion components if you want your ligation to work well
good luck, and welcome
Depending on the enzyme, you could try simply heating the tube to kill the enzyme, then doing the ligation, assuming the buffer conditions are similar enough; otherwise, do the EtOH pptn (but be prepared for losses). Why not split the expt into the two reactions? Digest the plasmid and run on a gel, then if you want to test your ligase, you could always get some marker, and try treating it with ligase, even 15 minutes at RT is enough. If your ligase is OK, you'll get a smear on a gel.
Well I guess I am testing the ligase but also the vector...so I want to know that when cut with those enzymes it can religate and the site is not effected.
I would definitely do at least a P:C:I and EtOH! Good luck!!
Thank you and have a super day