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).

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

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

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.