3 replies to this topic

[biochemical mayhem]

Hi cloners,

I have been facing a problem since two months. I have been trying to ligate the insert with one end cut with bamhI and filled up with Klenow, and the other end cut with ecori. The vector I have been using is cut with Ecori and SmaI. Ecori produce sticky ends, but SmaI produces blunt end.
I dont how compatible is the blunt end produced by klenow to ligate with the blunt end in the vector produced by SmaI.
The amount of the vector and the insert is okay. I always test small amount i.e 5 µl of the ligation reaction in agarose to confirm the ligated product. But I see nothing. I tried ligation in different temperatures and different buffer form different companies. But I think this is not the problem because I have similar experiment with other inserts and vector. The procedure worked well and I was successful in getting positive clones.
can anybody tell me what can be the possibilities to overcome this problem?
This has been haunting me every night...

[bob1]

How much vector are you ligating?  The total DNA content from both vector and insert should be less than 50 ng.

Are you sure your end-filling is working?  Could you blunt cut this end instead?

[phage434]

I would never spend that amount of time doing this.  Change strategies! For example, use PCR to amplify the plasmid backbone, adding whatever restriction site you want.  I think of blunting as a relic of the 80's, before we knew how to use PCR.

[biochemical mayhem]

Thanks guys
I have been using 100 ng of the total DNA with with insert:vector 3:1. How could I be sure about filling of the blunt end? Do you have strategy to analyse this? No, i couldnot do blunt end cutting of the insert because I have no other restriction sites, otherwise i have to start a new amplication with different restriction sites.
This time I am doing a single restriction of the vector and the insert with the same enzyme. Hopefully it will work. If it doesn't work, i will start again from amplification.

cheers!