Drop Dialysis of Ligations
Rich Kernan
dialyze a few drops of ligation reaction prior to electroporation to avoid
arcing. These filters are fairly expensive and i was wondering if anyone could
suggest an alternative or possibly a better method
Stephen P Lee
Would a cut piece of dialysis tubing work?
--
Stephen P. Lee Office: (604) 291-4291
Department of Biological Sciences Fax: (604) 291-3496
Simon Fraser University E-mail: Stephe...@sfu.ca
Burnaby, BC, Canada. V5A 1S6
Mark Siegal
using butanol to clean up ligations for electroporation.
Briefly,
1) inactivate ligase at 65C for 10 min
2) bring vol to 50 ul with dH2O
3) add 500 ul n-butanol
4) vortex 5 sec, then spin in microcentrifuge 10 min
5) discard supernatant, dry pellet
6) resuspend pellet in 10 ul dH2O and transform 5 ul
Works like a charm for me and others in this lab. The large volume of
butanol completely dehydrates the ligation reaction and reduces the ionic
strength. Arcing is never a problem and neither is loss of the sample.
Mark L. Siegal
Hartl Lab
Harvard University
Christian Fritze
Siegal) wrote:
> [butanol extraction method]
>
>
> Rich Kernan (KER...@UGA.CC.UGA.EDU) wrote:
> [microdialysis method]
Are these really necessary? I dilute a 20 uL ligation 1:5 in water, then
transform 2-3 uL by electroporation. It never arcs, and I get more
colonies than I know what to do with.
Christian
the End
Jim
J. Graham
Biology Department
Washington University of St. Louis
Mark Siegal
: How does a butanol extraction reduce ionic strength?
I was skeptical too at first, and still don't know how the salt gets
removed, but I did track down the following info. The butanol extraction
method for cleaning up ligations for electroporation is based on a method
for purifying oligonucleotides (NAR 19:674, 1991) after deprotection in
NH3 and lyophilization. The authors of that study compare n-butanol
extraction (same 10:1 ratio of butanol:aqueous) with Mg++/ethanol
precipitation, using UV/vis spectroscopy to assess the level of organic
contamination and conductivity measurements to assess the level of ionic
contamination. Mg++/ethanol precipitation reduced conductivity to about
30 percent of the lyophilized sample and butanol extraction reduced
conductivity to about 50 percent of the lyophilized sample. So somehow
the salt is removed.
Then again, if these numbers are reliable, diluting a ligation mix 1:1
with H2O reduces the ionic strength by the same amount as butanol
extraction!
Regards,
Mark
shyam...@gmail.com
> We frequently use 25 mm milipore filters floating in a petri dish of water to
> dialyze a few drops of ligation reaction prior to electroporation to avoid
> arcing. These filters are fairly expensive and i was wondering if anyone could
> suggest an alternative or possibly a better method
Shyamal