oligo purification

Jason E Stewart jasons at bmc.uu.se
Sat Mar 20 06:53:48 EST 1993


I can't help but making an analogy to help clarify my position for all
those who wrote in and advised to never check oligo's by gel as a waste of
time. 

If you go out to buy a half a dozen eggs, do you open the lid to see if per
chance any got broken in shipment? Or do you just grab the nearest carton
and go home to make a suffle for you dinner guests, only to arrive home and
find that 2 are in deed broken and you needed all 6 for the suffle. Do you
change the supermarket that you buy your eggs from? That doesn't help your
guest who now get macaroni because you couldn't take the 5 seconds to open
the lid. And what if the only other egg supplier is 5 kilometers away and
you don't own a car? 

Not all of us by oligos from big commercial suppliers, and need to make do
with the occassional sub-standard synthesis from the local facility. Having
taking a look at a number on oligos from two large facilities, one in CA
and one in TX, they too are subject to the bad day phenomenon. Some
techniques such as primer extension and PCR amplification from very few
starting copies in a large background of other DNA are very reliant on the
purity of oligos used.

Sure you can get it to work without purifying in most cases, but how much
extra time do you spend screwing with conditions compared to what you would
have had to do if you had purified?

The answer is: YOU DON'T HAVE THE FOGGIEST IDEA, BECAUSE YOU'VE NEVER
PURIFIED AN OLOGO AND DONE THE COMPARISON! But you still feel the need to
write in and give lousy advice to someone who doesn't know better. 

For those of you hard of reading, I will repeat once again. For most
applications you can get your oligos to work with a bit of parameter
manipulation (annealing time/temperature, buffer ion conc., addition of
detergents, etc.). 

But you can guarentee yourself a success if you do two things:1) Design
your oligos with a program made for that purpose, such as Oligo v4.0, and
2) Purify your oligo to make sure that 100% of the material you're using is
full length.

At the very least, run a gel to make sure that none of your eggs are
cracked.

Jason.

----------------------------------------------------------------
Jason Stewart                                 Father at large
Department of Medical Genetics            jasons at bmc.uu.se
Biomedical Center, Box 589                Tel 46 18 17 49 10
751 23 Uppsala                            Fax 46 18 52 48 69
Sweden



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