Protocol Online logo
Top : Forum Archives: : Molecular Cloning

how to improve ligation efficiency - (Nov/07/2005 )

how to improve the ligation efficiency? The vector is about 5000bp and the fragement is about
9000bp.We have no electrotransform.


What have you done so far?


I can not get the positive clone


QUOTE (Bomber @ Nov 8 2005, 08:39 PM)
What have you done so far?

You need to be more specific if you want constructive comment. What ligatio ratios have you done? Is it a sticky or blunt end ligation? What temperature did you do the ligation? Are you dephosphorylating the vector? etc etc


how are you transforming? i am assuming you are using chemically competent cells...are you making them or buying them?

what controls have you done and what is your data from those controls? that should help you pinpoint the problem


It is a stiky ligation. I digested the vector and fragment with AflII and XbaI. I did the ligation
with 22 for 3 hours and 4 overnight.I made the competent cell myself using Cacl2.It has been tested.


what is the profile of the ligation vs te control ligation and vs the unligated plasmid?


easily to ligated the 3000bp fragment into the vector.I had tesed the unligated plasmid,it digested well.