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Mysterious ligation...
Posted by: Marie (IP Hidden, New member, 3)
Date: June 20, 2006 01:30PM
Hi all of you!
First of all, i’m sorry for my english because i’m a francophone so i’ll make some mistakes!!! I’m working with students in an university and we have some problems having a successful ligation. Since the end of the session, I’m trying solving the problem. But, i’m still unable… I’m trying to ligate the plasmid vector pBlueScript 3000pb with insert GFP 750pb (of pUC18). I use Eco RI from Roche for the digestion and the vector is dephosphoryled with Shrimp Phosphatase alkaline, from Roche too. I’m doing the ligation with T4 ligase, always from Roche. After many failures, I’ve done electrophoresis in agarose gel to check the DNA. A big surprise was waiting for me! Before the ligation, the DNA is ok. I see the two bands. After the ligation, there’s no more DNA!! There are just few bands smaller than the insert… I don’t understand. It’s like the DNA is destroyed by something! I need some help because I don’t know what to do. Thank you! Marie Milette
Re: Mysterious ligation...
Posted by: charan (IP Hidden, Junior member, 14)
Date: June 21, 2006 11:01AM
Since you say that there is no DNA, I am tempted to suggest that you probably have a nuclease contamination in your one of your ligation reaction components and the small bands you see are probably degradation products.
You might want to try with a fresh ligase both the enzyme and buffer and make sure the water you use is free of nuclease. Good luck
Re: Mysterious ligation...
Posted by: Marie (IP Hidden, New member, 3)
Date: June 22, 2006 08:32AM
Thanks!
I have a new kit of ligase + buffers but i will try to "refresh" my water. Mayby it is contaminated. I'll try it next week! Marie
Re: Mysterious ligation...
Posted by: Marie (IP Hidden, New member, 3)
Date: June 28, 2006 03:46PM
OK.
I have tested my water and it's not the problem. I suspect my buffer or my ligase even if they're new... I will test them... Thanks! Marie
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