Cloning 180bp DNA in a vector
(by novalidaddress from nurfuerspam.de)
Sat Dec 18 17:31:09 EST 2010
if you consider the time, costs and effort to check lots of clones for
the correct sequence (specially when ligating 6 unpurified oligos),
I'd strongly suggest to order the whole construct, as the cost is
virtually nothing (<500 USD vs. the above (and the risk of failing/
wrong results from subsequent experiments), and you have someone to
blame if it does not work out. The goal of your work probably is not
to establish a procedure for homemaking 180bp inserts). See eg.
http://www.sloning.com and http://www.geneart.com as examples for a
companies offering gene synthesis services.
HTH & good luck!
On Dec 18, 10:29 pm, Pepa Florez Pérez <snipeur... from hotmail.com> wrote:
> Hello everyone!
> I must clone a 180 bp. DNA that I have designed in several vectors.
> Since it does not belong to a gene or cDNA I am not sure which one is the best method to carry out this project.
> I have thought of two possibilities:
> - to ask for 6 oligonucleotides, do the annealing and try to ligate them with a digested vector.
> - to get the DNA synthesized by a company, and subcloning by digestion.
> The problem is that I don't know which one is the proper approach, if there is any.
> About the oligos, we talked about not asking them purified by HPLC or PAGE since these are quite expensive procedures, but I am afraid that becuase of that I won't have a good annealing.
> And about the DNA synthesis... it cost around 200 euros/each... what we have considered as quite expensive too.
> Do you think that we will be successful working with not-purified oligos? Or something cheaper will become expensive late?
> Can you think of any other alternative?
> Thank you very much
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