ligation problem of double digested products - (Aug/01/2005 )

hi ,

i wanted to ligate two linear sticky ends framents (500bp, 600bp) to pET vector (3.6kb).
The amplified PCR product and vector restrected with RE NdeI and XhoI.

i checked if the restriction has done sucessfully or not, and it has done confirmly.
After resriction, i tried to ligate those two products.
But when i checked the ligated product on 0.8% agarose gel, i just could two band
exactly in corresponding to the vector size and inserts size.
i think the ligation hasn't worked.

So,i changed ligase to confirm if it is the problem of ligase,
but the result was just the same.

Then, to increase the efficiency of ligation, i perform CIAP treatment.
but Even after i treat CIAP, the result was just the same.

Is there anyone ever had the same problm as me?

HEEEEEEELP~~~~~~~~~~ me!

---

Hi,

well maybe your ligation has worked and you don´t see anything on your gel. I did the same thing a few weeks ago thinking that my ligation has not worked becuase I didin´t have the correct size in my gel. Anyway I transformed my ligation and guess what: I got colonies with insert of my correct size. So just try a few different rations and transform a few.

Good luck
Cheers

---

thank you for helping..

Today i tried to cofirm about ligation problem.
and definetly there were some problem.
becaus the result was quite strange.

i could see 8 kb size fragment on agarose gel (0.8%) in ligated products.
i don't know what it means.

In my guess, it is the product of 2 vectors.
i don't know how to solve the problem..

What i'm sure is i strictly kept the ratio that the protocol says. ...


please help me...

---

I find in general that looking at ligation products on a gel is simply frustrating, not illuminating. Just transform the mess and pick colonies. Pairs of vectors which ligate to one another don't seem to survive this process.

---