making a master mix for SDS-PAGE

[Michael L. Sullivan]

Since I mostly run 10% SDS-PAGE gels, is there any reason I can't make up a
master mix of acrylamide, water, SDS, and buffer that would need only to
have APS and TEMED added?

I know that such mixes are available commercially, but I wasn't sure
whether those might have some additive that would prevent some dire
consequence of mixing the other components for longer term storage and use.

Thanks for any insight.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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[clarosa]

I think it is not done this way because it does not work... otherwise,, we
would be doing it this way.

[Helen Niederwald]

In article <v04011700b62dcef7478a@[144.92.64.179]>,

mlsu...@facstaff.wisc.edu ("Michael L. Sullivan") wrote:
> Since I mostly run 10% SDS-PAGE gels, is there any reason I can't
make up a
> master mix of acrylamide, water, SDS, and buffer that would need only
to
> have APS and TEMED added?

I have been doing this for years. Works fine. I just keep it in the
fridge.
Usually lasts me a couple of weeks (4-8).

Helen.

>
> I know that such mixes are available commercially, but I wasn't sure
> whether those might have some additive that would prevent some dire
> consequence of mixing the other components for longer term storage
and use.
>
> Thanks for any insight.
>
> Mike
>
> Michael L. Sullivan, Ph.D
>
> U.S. Dairy Forage Research Center
> 1925 Linden Drive West
> Madison WI, 53706
>
> (608) 264-5144 Phone
> (608) 264-5147 Fax
>
> ---
>

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Before you buy.

[Michael L. Sullivan]

Maybe not. I just found the following in CSH press "Antibodies: A
laboratory Manual":

"For greter reproducibility from day to day, the acrylamide,
bis-acrylamide, buffer and water can be prepared in lagre batches and
either stored at 4 deg. C for 1 month or frozen in aliquots and used
indefinitely. Remove the required amount, warm to room temperature and add
ammonium persulfate and TEMED immediately before use."

I'd still be interested to know if anybody is doing this routinely. I sure
hate mixing up gel mix all the time!

Mike

>I think it is not done this way because it does not work... otherwise,, we
>would be doing it this way.
>

>"Michael L. Sullivan" wrote:
>
>> Since I mostly run 10% SDS-PAGE gels, is there any reason I can't make up a
>> master mix of acrylamide, water, SDS, and buffer that would need only to
>> have APS and TEMED added?
>>

>> I know that such mixes are available commercially, but I wasn't sure
>> whether those might have some additive that would prevent some dire
>> consequence of mixing the other components for longer term storage and use.
>>
>> Thanks for any insight.
>>
>> Mike
>>
>> Michael L. Sullivan, Ph.D
>>
>> U.S. Dairy Forage Research Center
>> 1925 Linden Drive West
>> Madison WI, 53706
>>
>> (608) 264-5144 Phone
>> (608) 264-5147 Fax
>>
>> ---

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[Tatiana Bibikova]

unsubscribe

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[Michelle Gleeson]

Many years back, when I did manual sequencing :-( , we routinely made
up batches of the buffer/water/acrylamide mix. It was kept for no
longer than one month, at 4degC, in a dark bottle wrapped in foil. We
let it warm up a bit, added the TEMED and APS, and poured. If it is
too cold when poured, it takes too long to set. Conversely, when you
have to make a fresh batch in a hurry, pouring even slightly warm
resulted in catastrophic premature setting. I don't know for sure
what would happen if SDS were added to the mix, but I imagine it might
precipitate out at cold temps. Thus tempting you to warm it up before
pouring... ;-)

Why not test it out? Not really much to lose.
Cheers,
Michelle

Molecular Parasitology Unit
University of Technology, Sydney
'don't cut too many corners, or you'll end up going around in circles'

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[engelbert...@my-deja.com]

In article <dd0a4d989b...@uts.edu.au>,

> I don't know for sure
> what would happen if SDS were added to the mix, but I imagine it might
> precipitate out at cold temps. Thus tempting you to warm it up before
> pouring... ;-)
>
> Why not test it out? Not really much to lose.

At the 0.1% pH 6.8/8.8 commonly used in SDS-PAGE SDS is soluble in the
cold. This would be different for acidic gels, however. As long as you
degass with ultrasound, the mix can be made with SDS. If you want to
degass with vacuum, you better add the SDS later, from a 10% stock
solution, as you'd get a mess otherwise (been there, done that).

My own procedure is slightly different, I have separate stock solutions
for buffer/SDS and acrylamide/bisacrylamide. That way I can use the most
appropriate acrylamide concentration for each problem, but still mix the
gels with a minimum of fuss. I add water to the solid acrylamide in the
manufacturers bottle, assuming that the amount in the bottle is as
stated on the label (I just want to minimize handling of that stuff).
Then I add the bis and freeze the solution in aliquots.

[Kevin A. Morano]

No problem. Works just fine, dark or light bottle. Good at least 2
months. I also use a 25% APS stock also good for months - don't make it
fresh. I am impatient, so I do a rapid polymerization:

10 ml gel mix (10%)
100 ul 25% APS
10 ul TEMED

Pour, overlay with water, come back 5 minutes later. Ready to go.

Kevin

[tfitz...@gilead.com]

>Michael L. Sullivan (mlsu...@facstaff.wisc.edu)
>Tue 07 Nov 2000 - 15:27:02 GMT>Since I mostly run 10% SDS-PAGE gels, is

there any reason I can't make up a
>master mix of acrylamide, water, SDS, and buffer that would need only to
>have APS and TEMED added?

>I know that such mixes are available commercially, but I wasn't sure
>whether those might have some additive that would prevent some dire
>consequence of mixing the other components for longer term storage and
use.

>Thanks for any insight.

>Michael L. Sullivan, Ph.D

We routinely prepare denaturing acrylamide mixes from commercial liquid
acrylamide:bis stocks, TBE and urea. The urea is dissolved by briefly
microwaving the mixture. We do not filter the stock solution in case the
liquid stocks contain that rumored gaseous stabilizer. At Boulder's
altitude (1 mile), degassing of acrylamide stocks is not necessary (if
there is reduced dissolved oxygen, there is probably not a lot of
stabilizer either). Stock is stored in polystyrene bottles on the benchtop
without foil wrapping for 2 months. At about 2 months, the polymerized
gels suddenly develop a UV shadow that tells us that it is time to prepare
a fresh stock. Nucleic acids eluted from the heavily shadowed gel behave
normally. Have not tried this with SDS, but expect that it will work.

Tim Fitzwater

Principal Research Associate

Gilead Sciences

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