Gene construction advice needed

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Jul 10 03:23:55 EST 2000


Hi Joe,
I think there is no such thing as an "Idiot´s guide to gene
construction". There seems to be a lot of know-how necessary to be
successful with such an approach. We have tried it years back, using 6
overlapping oligonucleotides of 50-60nt each. Even though the company
that made the oligos sent us an order form with 150 spaces to fill, they
actually weren´t able to make good 60mers. We tried other suppliers, and
the oligos were better, but after half a year of hard work and
approximately 2500$ for oligos we finally cancelled the project. 
Well, that was years back, and custom synthesis has significantly
improved since. If I had to do it now, I´d assemble the fragment in more
than one run. You say that any two oligos give perfect results, so I
would start assembling short fragments from two oligos first, eg. two
50mers that anneal with a 10-15bp overlap. The resulting fragment will
be around 90bp of dsDNA (after filling the gaps). Do the same for the
other side of the wanted fragment, and so on until you have overlapping
fragments of dsDNA that cover the whole wanted fragment (overlaps no
smaller than 15bp). Then, mix the fragments in equimolar amounts,
denature by boiling, slowly cool to room temperature, add dNTPs and
Klenow or T4 polymerase, and incubate for 30min. Then, take a small
aliquot of the reaction and perform a PCR reaction with two extra
primers on the 3´ and 5´ end of the wanted (full length) fragment. 

Hope this helps,
Frank


"Joseph C. Bagshaw" wrote:
> 
> I'm looking for advice on a gene construction project.  I'm trying to
> assemble a 219 bp gene from oligonucleotides.  I designed the oligos so
> that each overlaps the next by ten complementary bases.  The plan then is
> to fill the gaps, then ligate.  I can get perfect results with any two
> adjacent oligos, but when I try to assemble the whole, instead of a 219 bp
> band I get a smear.  Any advice or direction to any source of information
> would be greatly appreciated.  I have not found any reference or "how
> to" source, like "Idiot's Guide to Gene Construction".  Please mail to the
> address below or post here.
> 
> ********************  HAVE GENES, WILL TRAVEL  ********************
> Joe Bagshaw, Worcester Polytechnic Institute
> jbagshaw at wpi.edu
> Roadkill on the information superhighway.






More information about the Methods mailing list