how to move for ligation with low conentration of digested plasmid - (Apr/27/2006 )
I am trying to clone in pGBKT7 vector but the vector concentration is too low. I proceeded for a double digestion using EcoRI and BamHI sites but now i am confused wheather I shall gel elute or ethanol precipitate or follow any other method to remove restriction enzymes from the mixture and proceed for ligation with minimum loss of DNA. Please suggest me.
The problem is if you perform ethanol precipitation you also precipitate the enzymes... so you should heat inactivate them or you could clean up your digestion with a pcr clean up kit.
By the way, what is the concentration of your vector?
i agree with fedex. A kit is more reliable to purify. Then elute in a great volume (100µl and pellet with adding 1ml buthanol and vortex well. wash twice with 1ml etoh70% and resuspend in 10µl