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I haven't done TA cloning in a very long time. Typically, TA cloning is done with blunt-ended DNA fragments that have been modified by terminal transferase. Normally, a blunt-ended vector digest is treated with terminal transferase and TTP. (If the vector is cut by non-blunt-ended restriction endonucleases, it must be filled in with Klenow or equivalent before adding the T. PCR fragments must be treated with terminal transferase and ATP, or if using Taq (not recommended these days with high-fidelity polymerases available) a significant fraction of PCR products will already have the A overhang. Ligation will result in the insert being inserted (supposedly) randomly in forward and reverse orientations. We don't do this method anymore because it is too problematic and too time-consuming. A double digest with two different overhangs (both vector and PCR product) is much preferred, and results in a high percentage of recombinants with the correct orientation. We usually overdigest PCR products and vectors for at least 3 hr to ensure double-cutting. Phosphatase treatment of the vector after double digest prevents re-ligation of empty plasmid that has only been cleaved by one enzyme. RE digestion of PCR products requires at least 3 additional nucleotides in front of the recognition site. We usually add TGC in front of the recognition site for all PCR primers. Unfortunately in your case, NheI and BamHI have the same 3' C overhang, so double digestion with these two enzymes will result in a random orientation of your PCR product in the vector. I would recommend using a different RE for one of the sites to ensure correct orientation of the PCR product when ligated. We routinely use NdeI and PstI for most of our expression plasmid constructs (assuming these recognition sites are not present in our vector outside the cloning region--this is typically a good pair for pET vectors, and is a good double digest), as NdeI has a start codon within it. (NcoI is another possibility, if your first codon after the start begins with "G"). For details, see our lab wiki with time-tested methods: http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Protein+Engineering+Protocols Cheers, -- ------------------------------------------------------------------------ Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask] Raji Edayathumangalam wrote: > Hi Vijay, > > I have heard of TOPO-TA cloning. Not sure what T/A cloning is. > > I have a couple to check based on your description: > 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a > 'vector only' transformation control to determine how many colonies are obtained in the > 'vector+insert' plate above background levels. > 2) For NheI/BamHI, a sequential digestion (rather than a double digestion) is recommended. > 3) Make sure the primers have sufficient extensions (6-8nt) outside of the RE site. > 4) Make sure both enzymes work by doing single digestion controls with the vector. It's obviously > hard to tell with the PCRT product > 4) Titrate vector:insert ratios -- lower and higher than you mention > 5)....... > > Once ALL possibilities are exhausted and when nothing else works, I have seen people reorder the > exact same primers and then things have worked like a charm! > > Hope that helps. > Raji > > > > > ---------Included Message---------- > >> Date: 1-sep-2008 03:06:29 -0400 >> From: "vijay srivastava" <[log in to unmask]> >> Reply-To: <[log in to unmask]> >> To: <[log in to unmask]> >> Subject: [ccp4bb] regarding cloning >> >> Hi, >> I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The >> > restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. > I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion > and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is > 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone > through double digestion i am not getting my insert at the correct position.Some time in the gel > only the size of the vector was there. > >> Connect with friends all over the world. Get Yahoo! India Messenger at >> > http://in.messenger.yahoo.com/?wm=n/ > > ---------End of Included Message---------- >