Why the ligation does not work? - (Jul/27/2007 )

I am trying to put a 2Kb insert in to p3XFlag-CMV-14 vector. I use EcoRI and KpnI to modify the insert and vector. Before that I have checked the insert sequence to make sure that it does not contain EcoRI and KpnI digestion site. When do ligation, I set a vector control and use different ration of insert and vector. After transformation, on the control plate, there are none to few colonies and there are many colonies on the experiment plates. However after I pick up 10 colonies for mini and digestion, none of them have right size insert, either no insert or strange digestion pattern.

Could anyone help me to figure out the problem?

Thank U.

Maybe you can check your ligation buffer. Does it contain ATP? What kind of ligation buffer did u use?
And I think you also should check whether both enzymes work or not?

Lucy

Did you run the original plasmid and your miniprep plasmid uncut side by side. You should be able to see a difference in size.

What do you mean by strange digestion pattern. Is it possible that you are having some star activity of EcoR1 while digesting the miniprep?

How about your ligation condition? Try to pick some colonies for direct PCR/colony PCR.

Star activity is unspecific enzyme cutting on your nucleotides. Usually occur if you put too much enzyme or longer incubation time.

Ligation MIGHT not work even if you got colonies, maybe some self ligated plasmid that being transformed inside? Good luck!

Is your insert a PCR product? If so, double-check the primers that you used. Make sure the restriction enzyme cut site is at the correct end of the primer. If it's at the wrong end, the PCR will still work, but your insert will not be cut properly by both enzymes.