using agar-agar

[JonathanCline]

I'm making gels using agar-agar purchased from an indian grocery store
(curiously, it's a chinese product yet unavailable at an asian grocery
store; the asian groceries only had pre-sweetened agar powders). How
do I measure agar-agar for reliable gel concentrations (for repeatable
results), anyone?

The e-z protocol is detailed in MIT Bioengineering 20.20,
http://openwetware.org/wiki/2020(S09)_Lecture:week_4

However, it says use a tablespoon of melted agar-agar which I assume
varies considerably. Should I dilute smaller amts until I still end
up with the lightest gel to yield best results or what?

Eventually I'll switch to standards, for now I'm trying what's on
hand.

## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

[Josh Perfetto]

If I am reading the same thing you are, it doesn't say a tablespoon of
melted agar-agar, but rather "Melt 1/2 tablespoon agar-agar with 1/2 cup
running buffer..." The point I'm making is not the typo of 1/2 tablespoon
vs 1 tablespoon, but that you are measuring the solid agar, and so it
shouldn't vary that considerably. If you want to be more precise though,
you would measure the agar by mass and not volume.

What you really want to do for reproducability is keep the percent agar
(versus buffer) the same for similar gels. It's most common to measure this
percentage as w/v (weight agar / volume buffer). You can then vary the
percentage agar of your gels based on the length of DNA you are trying to
separate. For example, with pure agarose, you would use a 0.3% gel to get a
good separation for molecules 5-60 kb, 0.9% for 0.5-7 kb, 2.0% for 0.1-3 kb,
etc. I don't know exactly what percentages would be best for agar (this is
something we should measure and publish unless someone can dig something
up), but I suspect they would be similar.

BTW those percentages I gave are for grams of solute (agar in this case) per
100 ml of solvent, as is typical for w/v percentages. I could never deal
with measuring things in tablespoons or cups :)

So if you are measuring your agar by mass, and buffer by volume, the only
thing you would really need to worry about is boiling away buffer when
melting the gel (thus increasing your concentration), or variability in the
composition of the agar (since it is not purified agarose). But since you
normally run a ladder lane, it's not like you have to be totally exact, you
just want to get the gel percentage roughly right for what you're trying to
separate. What you're doing when you adjust the gel concentration is
adjusting the pore size of the gel, you don't want it to be too big that all
the DNA molecules pass right through, or too small that they all get stuck.

-Josh

[Tito Jankowski]

Hey Jonathan,

Great to hear you're trying this on your own! Common Agar-agar is ~99%
agar seaweed. As long as it's in powder form, your results would be
fairly consistent if you used a tablespoon each time. If you're using
anything but powder, making consistent gels is more difficult. Mixing
up a big batch at once and using that throughout multiple experiments
is another way to get consistency.

This is what my agar-agar looks like.

Everyone, what does YOUR agar-agar look like? Post pics!

Tito

[Aaron Rodriguez]

Tito, I got the exact same baggies from my local oriental store!   

-AR

Aaron Rodriguez
(c) 301 254 1718

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[JonathanCline]

Thanks Josh. Part of the confusion is that my agar-agar is not
powder, it is stick form (8" sticks similar to long rice noodles),
like in this picture:

http://tbn0.google.com/images?q=tbn:H4yk5_hmMllwVM:http://img.quamut.com/chart/7711/05_agaragar.jpg

I'll upload better pics later. I guess others are using powders.

A volume measurement would only make sense if I chopped it up. By
weight, it's possible. In discussion w/ Tito he also suggested
using a loading dye which would show during the run, so I won't run my
DNA out the end of the gel. And also mix up a big batch so it's
consistent across multiple gel pours. (I haven't recv'd a ladder
yet.)

[Daniel Wexler]

Most natural foods stores sell agar in powder form.

[Frankco]

Hi Tito,

What about Micropropagation grade agar? You can get 500 grams from Caissons
Labs for $39 or if you don't want that much you can get 56 grams food grade
agar for $4.20 from Amazon. Both are in powder form and easy to work with.

http://www.caissonlabs.com/catalog/view_product/41

http://www.amazon.com/NOW-Foods-Agar-Powder-oz/dp/B000MGSJ5A

Frank

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>
>
>
> On Feb 27, 2009, at 8:57 PM, JonathanCline wrote:
>
>>

[Tito Jankowski]

Both look good to me!

Tito

[JonathanCline]

On Feb 28, 9:26 am, "Frankco" <f...@frankandjackie.com> wrote:
> Hi Tito,
>
> What about Micropropagation grade agar? You can get 500 grams from Caissons
> Labs for $39 or if you don't want that much you can get 56 grams food grade
> agar for $4.20 from Amazon. Both are in powder form and easy to work with.

Are the lab-grade agars edible? I'm curious. Would be interesting
to add to the list of audience-gasps-when-Mac-swallows-it protocols if
using all edible materials.

The food grade agar obviously is very edible as this yummy googled
recipe shows..

Agar Agar Dessert

Agar Agar powder is a great substitute for gelatine. Its advantage is
that it will gel while warm, and does not have to be refrigerated to
set. A dessert can be made and ready to serve in a about 15 minutes.
It is also good for vegetarians as unlike gelatine which is made from
animal products, Agar Agar is made from a vegetable source.

Ingredients

375 mls water
375 mls evaporated milk
1/2 cup sugar
2 tsp Agar Agar powder
Almond essence
Kiwi fruit, Strawberries.

[Frankco]

There is also gellam gum which hardens crystal clear. It's a bit more $45
per 500 grams. Not sure how that would work.

>Everyone, what does YOUR agar-agar look like? Post pics!

Here's my agar.

[Nathan McCorkle]

I've been using the Telephone brand Agar for months of culture plate work. Seems good enough, haven't tried electrophoresis with it though.

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics