Bio-Rad Acrylamide Solutions and Powders User Manual

Initiators

Chemical polymerization is initiated by
Ammonium Persulfate and TEMED. They are reactive
by nature and may be prone to oxidation or decomposi­tion. APS can be stored desiccated at room temperature
for at least 1 year. Solutions should be made fresh daily,
since persulfate in solution decomposes rapidly.
TEMED can be stored tightly closed and desiccated
either at 4 °C or at room temperature for at least 6
months. After 10 to 12 months, there is a significant
reduction in activity and an increase in the amount
required for polymerization. If refrigerated, TEMED
should be warmed to room temperature before opening
to avoid the release of condensation into solution.

Temperature and Oxygen

Oxygen is an inhibitor of free radical polymeriza­tion. Proper degassing of the gel solutions immediately
prior to pouring the gel is therefore critical for repro­ducibility. To insure faster and more complete
degassing, bring the gel solution to room temperature
first, as cold solutions have a greater capacity for dis­solved oxygen. Pouring a gel with a cold solution will
have a substantial negative effect on the rate of polymer­ization and on the quality of the resulting gel as well.

Contaminants in Buffers

Contaminants in buffer reagents, gel additives, and
laboratory water can inhibit polymerization. The most
common contaminants of these reagents are metals,
non-ion buffers, and breakdown products. Partially
inhibited polymerization will result in gels with greater
porosity than intended, and the molecules will have
greater mobilities.

For a more detailed discussion of gel polymeriza­tion, request bulletin 1156 from your Bio-Rad
Representative, or in the U.S. call 1-800-4BIORAD.

Section 5
Risk and Safety Information

Hazardous Acrylamide
components N-N’-Methylene-bis-acrylamide

Risk and May cause cancer. May cause heritable
safety phrases genetic damage. Toxic in contact with skin

Safety symbol
CAS number 79-06-1 (Acrylamide)

RID/ADR number 6.1/12c UN 2074
RTECS number AS 3325000
EINECS number 2011737
WGK number 3

and if swallowed. Danger of serious dam­age to health by prolonged exposure. Avoid
exposure - obtain special instructions before
use. If you feel unwell, seek medical advice
and show label where possible.

Kann Krebs erzeugen. Kann vererbbare
Schäden verursachen. Giftig bei Berührung
mit der Haut und beim Verschlucken. Bei
längerer Exposition Gefahr ernster Gesund­heitsschäden. Exposition vermeiden - vor
Gebrauch besondere Anweisungen ein­holen. Bei Unwohlsein ärztlichen Rat ein­holen. Wenn möglich dieses Etikett
vorzeigen.

Peut causer le cancer. Peut causer des
altérations génétiques héréditares. Toxique
par contact avec la peau et par ingestion.
Risque d´effets graves pour la santé en cas
d´exposition prolongée. Eviter l´exposition ­se procurer des instructions spéciales avant
l´utilisation. En cas de malaise consulter un
médecin (si possible lui montrer l´étiquette).

Può provocare il cancro. Può provocare
alterazioni genetiche ereditarie. Tossico a
contatto con la pelle e per ingestione. Peri­colo di gravi danni per la salute in caso di
esposizione prolungata. Evitare l´espo­sizione - procurarsi speciali istruzioni prima
dell´uso. In caso di malessere consultare il
medico (se possibile, mostrargli l´etichetta).

h

110-26-9 (N-N’-Methylene-bis-acrylamide)

Premixed Preweighed

Acrylamide/Bis

19:1, 30 g Catalog Number 161-0120
19:1, 150 g Catalog Number 161-0123
29:1, 30 g Catalog Number 161-0121
29:1, 150 g Catalog Number 161-0124

37.5:1, 30 g Catalog Number 161-0122

37.5:1, 150 g Catalog Number 161-0125

Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547

4

LIT306 Rev C

Table of Contents

Section 1 Preparation Instructions ..........1

Section 2 Protocols for Sequencing

Gel Preparation..........................2

2.1 Stock Solutions ..................................2

2.2 Gel Preparation ..................................2

Section 3 Protocols for Laemmli

Gel Preparation..........................2

3.1 Stock Solutions ..................................2

3.2 Gel Preparation ..................................3

Section 4 General Recommendations ......4

Section 5 Risk and Safety Information ....5

Section 1
Preparation Instructions

For the following %T* stock solutions, add dis­tilled, deionized water in the given amounts and stir for
30 minutes. Solutions should be mixed directly in the
bottle to avoid contact with Acrylamide and Bis.

For the 30 g bottle: For the 150 g bottle:

%T add H2O final volume add H2O final volume

50 33 ml 60 ml 162 ml 300 ml
40 48 ml 75 ml 237 ml 375 ml
30 73 ml 100 ml 362 ml 500 ml

Filtering is not required but is recommended for
the highest quality electrophoresis results.

Recommended shelf life for the stock solution is 30
days at 4 °C.

*%T and %C

Polyacrylamide gels are described by reference to
two characteristics:

1) The total monomer concentration (%T):

g Acrylamide + g Bis-Acrylamide

%T=

2) The crosslinking monomer concentration (%C):
%C=

In mixing solutions, a 30% w/v stock solution cor-

responds to 30% T. The %T will vary depending on the
amount of water added. The %C remains constant at all
dilutions.

†Laemmli, U. K.,

total volume

g Bis-Acrylamide

g Acrylamide + g Bis-Acrylamide

Nature,

227, 680 (1970).

x 100

x 100

Section 2
Protocols for Sequencing
Gel Preparation

2.1 Sequencing Gel Stock Solutions

Stock Solutions

A. Bio-Rad Preweighed Acrylamide/Bis 19:1.30%

Solution:

Follow instructions above for mixing and storing.

B. 10x TBE Buffer, 0.89 M Tris base, 0.89 M Boric

acid, 20 mM EDTA:

108 g Tris base
55 g boric acid

9.3 g Na2EDTA-H2O
Bring up to 1 liter in deionized water. The pH of
this solution should be 8.3. Do not add acid or base
to alter the pH. Alternatively, use Bio-Rad’s
Premixed TBE Buffer.

C. 25% Ammonium Persulfate (APS):

Dissolve 0.25 g in 1 ml distilled water. Make

fresh daily.

D. TEMED:

Use neat from the bottle.

2.2 Sequencing Gel Preparation

4% 5% 6% X%

30% Acrylamide/Bis 19:1 20 ml 25 ml 30 ml 5(X%)=ml
10x TBE 15 ml 15 ml 15 ml 15 ml
Urea 63 g 63 g 63 g 63 g
TEMED 150 µl 150 µl 150 µl 150 µl

25% APS 150 µl 150 µl 150 µl 150 µl

Adjust the volume to 150 ml with distilled deion-

ized water. Degas before polymerization.

Section 3
Protocols for Laemmli
Gel Preparation

3.1 Laemmli Gel Stock Solutions

A. Bio-Rad Preweighed Acrylamide/Bis, 30%

solution: Follow instructions above for mixing and

storing.

B. 1.5 M Tris-HCl, pH 8.8:

54.5 g Tris base
~150 ml distilled water
Adjust to pH 8.8 with 1 N HCl. Bring volume up to
300 ml with distilled water and store at 4 °C.

C. 0.5 M Tris-HCl, pH 6.8:

6.0 g Tris base
~60 ml distilled water
Adjust to pH 6.8 with 1 N HCl. Bring volume up to
100 ml with distilled water and store at 4 °C.

D. 10% SDS (w/v):

Dissolve 10.0 g SDS in distilled water with gentle
stirring and bring up to 100 ml. Store at room tem­perature.

E. 10% Ammonium Persulfate (APS):

Dissolve 100 mg APS in 1 ml distilled water.

Make fresh daily.

F. TEMED:

Use neat from the bottle.

3.2 Laemmli Gel Preparation

Stack Resolving Gel

4% 7.5% 12% X%

30% Acrylamide/Bis 37.5:1 3.3 ml 25 ml 40 ml 3.3(X%)

0.5 M Tris-HCl, pH 6.8 6.3 ml -- -- --

1.5 M Tris-HCl, pH 8.8 -- 25 ml 25 ml 25 ml
10% SDS 250 µl 1.0 ml 1.0 ml 1.0 ml
Distilled deionized water 15 ml 48.5 ml 33.5 ml 73.5-(A)*
TEMED 25 µl 50 µl 50 µl 50 µl
10% APS 125 µl 500 µl 500 µl 500 µl

25 ml 100 ml 100 ml 100 ml

=(A)*ml

Degas before polymerization

* The letter A designates the volume of 30% Acrylamide/Bis 37.5:1

required to produce the specified percent gel (X%).

Section 4
General Recommendations

Acrylamide and Bis

These electrophoresis purity reagents can be stored
away from direct sunlight, dry, at room temperature
(23-25 °C) for at least 1 year. Heat and light may cause
autopolymerization. Monomer stock solutions contain­ing Acrylamide and Bis should be stored at 4 °C for no
longer than 1 month. During prolonged storage, hydrol­ysis of Acrylamide to acrylic acid will occur.

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