Instruction Manual

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 tem-
perature.
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%)
=(A)*ml
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
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 contain-
ing Acrylamide and Bis should be stored at 4 °C for no
longer than 1 month. During prolonged storage, hydrol-
ysis of Acrylamide to acrylic acid will occur.
3
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 Na
2
EDTA-H
2
O
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.
2
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 dis-
tilled, 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 H
2
O final volume add H
2
O 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):
%T=
g Acrylamide + g Bis-Acrylamide
x 100
total volume
2) The crosslinking monomer concentration (%C):
%C=
g Bis-Acrylamide
x 100
g Acrylamide + g Bis-Acrylamide
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.
1
†Laemmli, U. K., Nature, 227, 680 (1970).
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