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: [EMAIL PROTECTED]
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" <[EMAIL PROTECTED]>
Reply-To: <[EMAIL PROTECTED]>
To: <[email protected]>
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.
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