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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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ligation problems
Posted by: Stefanie (IP Hidden, New member, 2)
Date: July 10, 2006 03:10AM
Hi all of you!
My name is Stefanie and I am having some problems with a ligation I've been trying lately. I'm trying to ligate a 1550bp insert into a 12000bp vector. The insert has been cut out of another vector using the restriction enzyme Bgl2. The vector has been cut with BamH1. Bgl2 and BamH1 fragments can be ligated to one another (Bgl2 and BamH1 create the same sticky ends). One should expect that the insert can be ligated in both directions in the vector. But what I'm seeing is that the insert is always ligated in only just one direction. And of course, the result that I'm waiting for is for the insert to be in the opposite way. Is this a common problem? And what can I do to influence the direction in which the insert ligates into the vector? Kind regards, Stefanie
Re: ligation problems
Posted by: Stefanie (IP Hidden, New member, 2)
Date: July 11, 2006 11:25PM
Hi,
Could it be that when the insert ligates in the opposite way, the vector produces something that is toxic to the E.coli host strain? And that this is not the case when it ligates in the other way? So all the colonies which have been transformed with the vector with the insert in the opposite way all die and in this way stay undetected? And would it be possible to avoid this by using another host strain?
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