ligation problem - (Apr/21/2008 )
i am going to do ligation in pBI 121 vector, i digested both vector and my fragments(isolated by pcr with primers with R.E sites) with same emzymes,so they should form sticky ends,but i donot know where is the problem,i am not getting ligated product,is there any need to do dephosphrelation of any one product or not.please help me in this issue.
Thanks in advance.
[quote name='ramirddyag' date='Apr 20 2008, 11:14 PM' post='133280']
i am sorry its phophorelation.
What ratio are you using? Perhaps you are not using enough samples?
You don't need to phosphorylate the insert. There should be 5' P after RE digestion.
What do you mean you are not getting the ligated product??
I presume you are transforming bacteria with your product? Do you mean you get no colonies? or no correct colonies? Are there any colonies on your control plate?