Cloning PCR fragment into the expression vector

Jayakumar R jakku71 at yahoo.com
Thu Mar 7 12:25:10 EST 2002


the agarose gel is the problem here.  Dont do all that. the PCR product is
clean enoughto do the ligation with.  Run it through an agarose gel and you
get sulphate contamination. However low sulphates are in the eluate, ligases
still show inhibition.  You can try shot gun cloning or use a commercial kit
for cleaning the PCR product.  you can try quiagen or invitrogen.  I used to
just take some of the PCR product and ligate  into the EcoRV site of my
clone and get lots of clones.  Anyway, the cardinal rule is try to avoid the
agarose step as much as possible in any ligation work.  Remember whenever
you elute the DNA from an agarose gel, whatever you do, there will always be
traces of sulphates in the DNA obtained, which is enough to inhibit the
ligase.  But blunt end ligation is quite easy when you use the PCR product
as such. 
    I would also suggest use of T-tailed vectors like the pGEMT-easy vector
sold by promega to cone PCR amplicons.  I used to use this for all my PCR
ligations and it works like a wonder every time.
    best of luck
sincerely
jayakumar

> From: Joyce <mdccsw at nus.edu.sg>
> Organization: mail2news at nym.alias.net
> Reply-To: mdccsw at nus.edu.sg
> Date: 5 Mar 2002 08:34:40 -0000
> To: methods at hgmp.mrc.ac.uk
> Subject: Cloning PCR fragment into the expression vector
> 
> Dear Colleagues,
> 
> Need help. I am doing some blunt end ligation to clone a PCR product (~700
> bp) into an expression plasmid (~4.9 kb), but couldn't get it work.  can
> anyone give any suggestion?
> 
> For the PCR product, I have purify through agarose gel to get rid of Taq
> polymerase before doing ligation while the vector, a double digestion was
> performed with EcoRI and XhoI and Klenow fragment + dNTP was added to fill
> the 5' overhang. Then the 4.9 kb band was purified from agarose gel and
> performed dephosphorylation with Alkaline phosphotase to avoid self
> ligation. The linearised vector was again purified from agarose gel to get
> rid the Alkaline phosphatase (which will inhibit ligation). Ligation process
> was performed  before transformation was done.
> 
> 
> <http://www.biowww.net/forum/read.php?f=1&i=5337&t=5337>


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