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" <[EMAIL PROTECTED]> >Reply-To: <[EMAIL PROTECTED]> >To: <CCP4BB@JISCMAIL.AC.UK> >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----------
