Gene construction advice needed

Dr. Duncan Clark drc at nospam.demon.co.uk
Mon Jul 10 03:34:23 EST 2000


In article <Pine.OSF.4.21.0007080752270.29499-100000 at reno.WPI.EDU>,
Joseph C. Bagshaw <jbagshaw at wpi.edu> writes
>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. 

Easy with PCR:

Synthesise first pair of oligos with overlapping 10-14bp adjusting to
make sure Tm is not too low i.e. >40C.

PCR using both primers, Pfu and 15 cycles max. in 100ul volume.

Remove 1ul and re-PCR with 5' primer and a new 3' primer that overlaps
the first duplex by 10-14bp as before. Repeat ad infinitum until you get
to your final product. We've done this for I think four constructs up to
maybe 400bp. It can get a little tricky towards the end and on one I had
to gel purify 300bp or so fragment just to be able to PCR the next one
along.

Finally clone and sequence. You must sequence. We do get the odd clone
with missed single bp or changed bp, particularly in the 5' primer which
suggests primer synthesis oddities by our commercial supplier.



1.      5'..........>
                 <...........3'         PCR1

2.      5'..........>
          |..........
                  <...........3'        PCR2

3.      5'..........>
          |...................|
          |...................|
                          <..............3'     PCR3 

4.      etc. etc.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






More information about the Methods mailing list