ligation issues

[Jess]

Message: 2
Date: Fri, 29 Jun 2007 14:16:10 -0700 (PDT)
From: Grace Colletti 
Subject: Cloning Problems
To: methods from magpie.bio.indiana.edu
Message-ID: <685037.82936.qm from web34711.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

I am trying to clone a 3.2 insert into a 5.3 kb vector (pTRE2hyg Clontech). I've linearized my vector by cutting the MCS with BamHI & NheI. I cut out my insert with BamHI and XbaI. I've tried both CIPing the vector and not (since BamHI and NheI don't create compatible overhangs, I don't think CIPing is necessary??). I ligate my gel purified products using T4 ligase. I've tried ligating for 10 minutes, 20 minutes, and 1 hr. I've done this several times and still I only get vector without insert colonies...does anyone have any suggestions?


everyone else had great suggestions, but I have a couple that were not listed...
   
  Generally what I do is adjust my DNA concentration to 30 fm ends (vector) and 90 fm ends (insert), calculation based on ng/uL concentration of DNA and bp DNA. I ligate (10 uL total reaction volume) for 18-24 hours at 4C. 
   
  If you are worried about transformation efficiency, try the protocol given in the Stratagene mutagenesis kit for transformation, involves B-mercaptoethanol etc. (If you can't find it, email me off list and I will send you the protocol.) I have used this with different competent cells prepared all kinds of ways. This works well if you are having trouble due to low efficiency.
   
  Even if my ends are not compatible, I still Ciap treat my vector. And ditto on the "did you put enough extra bases on the ends of your primers to get good cuts from your enzymes" If not, i have a quick fix for you (without ordering new primers) - I just sorted this problem out for another fella in my lab a few days ago. just do a quick tailing reaction with Taq and ligate into pGEM-T (or any linearized T-vector) then you can transform, miniprep and digest your insert out (gel purify). This has the added bonus of knowing that you are getting complete digestion, which can be worrisome with some double digestion reactions.
   
  Not to be repetititve, but what sounds like to me is that you're not getting complete vector digestion, which is why you're getting so much vector religation, with or without CIAP. I would try to sequentially digest, or better yet, find someone in your lab- (or in your lab stock), that has your vector with gene insert in the same sites, and use that to digest your vector. This way, you can assess digestion, and remove that problem from your list of issues.
   
  Frankly, and not to say anything bad about certain companies, but we have noticed recently in our building- not just in our lab, that ligations have been getting harder recently, more touchy. We're having to just be anal retentive about everything, gel purifying our digests- inserts and vectors, sequentially digesting instead of doubling and eluting with pH 8 water instead of EB/Tris. I don't know what company your T4 comes from- but I hope you'll let me know what works, and maybe whose T4 you're using. If its the same as ours, I will pass on what has worked specifically for the T4 we are using.
   
  Cheers and I hope this was helpful.
   
   


    Jess
   
  Grad. Res. Asst.
  University of Texas- Austin
  Institute for Cellular and Molecular Biology
  Department of Medicinal Chemistry
             -Hook 'em Horns!
   



       
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