Polyacrylamide Gel Electrophoresis
(PAGE)
Gel electrophoresis remains to be a technology in modern-day bioanalytics which cannot be replaced by any other. In the vast majority of cas, Vertical gels are produced from acrylamide which with bis-acrylamide and after the addition of ammonium persulphate (radical donator) and TEMED (catalyst) form a very fine and extremely constant network by means of a radical chain reaction.
Application, however, is made more difficult as acrylamide is a very strong nerve poison, the carcinogenic and mutating properties of which have been clearly proven in animal testing. Acrylamide is primarily resorbed through the skin, but above all, it is the absorption of dust in the respiratory tracts or through the facial mucous membrane when weighing out the powder which caus the most rious problems. Rotiphore® ready-to-u solutions provide the perfect remedy. The solutions, which have been stringently controlled with regard to their acrylamide content and pH, are significantly less dangerous in their application.
They are subquently very easy to u and enable reproducible and high-resolution gel electrophor
esis. In our Roth range you will also find all additional reagents needed, such as TEMED, APS, SDS or gel electrophoresis buffers which form a perfect, coordinated team (e below).
Rotiphore® Gel Solutions
Ready-to-u acrylamide/bisacrylamide mixtures
Rotiphore® Gel 30 (37.5:1): 30 % acrylamide/bisacrylamide, mixing ratio 37.5:1.
Ord.-No. 3029.1 (1 l), 3029.2 (250 ml)
Rotiphore® Gel 40 (19:1): 40 % acrylamide/bisacrylamide, mixing ratio 19:1.
Ord.-No. 3030.1 (1 l)
Rotiphore® Gel 40 (29:1): 40 % acrylamide/bisacrylamide, mixing ratio 29:1.
Ord.-No. A515.1 (1 l)
Rotiphore® Gel 40 (37.5:1): 40 % acrylamide/bisacrylamide, mixing ratio 37.5:1.
Ord.-No. T802.1 (1 l)
Acrylamide- and bisacrylamide solution, ready-to-mix
Rotiphore® Gel A: 30 % acrylamide solution. Ord.-No. 3037.1 (1 l)
Rotiphore® Gel B: 2 % bisacrylamide solution. Ord.-No. 3039.1 (1 l), 3039.2 (250 ml)
Acrylamide/bisacrylamide mixtures for automated quencing (fluorescence free) Rotiphore ® NF-acrylamide/bis- solution 40 % (19:1): ready-to-u 40 %
acrylamide/bisacrylamide, mixing ratio 19:1. Ord. No. A516.1 (250 ml)
结果英文Rotiphore ® NF-acrylamide/bis- solution 40 % (29:1): ready-to-u 40 %
acrylamide/bisacrylamide, mixing ratio 29:1. Ord. No. A121.1 (250 ml)
Rotiphore ® NF-acrylamide/ bis- solution 30 % (29:1): ready-to-u 30 %
acrylamide/bisacrylamide, mixing ratio 29:1. Ord. No. A124.1 (250 ml), A124.2 (1 l)
Ready-to-u quencing gel solutions
Rotiphore ® quencing gel concentrate: 25 % acrylamide/bisacrylamide, mixing ratio 19:1
and 50 % urea. Ord. No. 3043.1 (1 l), 3043.2 (100 ml)
Rotiphore ® DNA quencing system (1 l quencing gel concentrate, 1 l quencing gel
diluent, 250 ml quencing gel buffer) Ord. No. A431.1 (1 Kit)
Recommended Applications: Separation of Recommended gel solution
% C Acrylamide / bisacrylamide
Gel 40 (3030.1)
5 19:1 NF-acrylamide/bis-solution 40 % (A516.1) 5 19:1 Nucleic acids
Sequencing gel concentrate 25 % (3043.1) 5 19:1 Gel 40 (A515.1)
3.3 29:1 NF-acrylamide/bis-solution 40 % (A121.1) 3.3 29:1 Nucleic acids and
proteins NF-acrylamide/bis-solution 30 % (A124.1) 3.3 29:1 Gel 30 (3029.1) 2.6 37.5:1 Proteins
Gel 40 (T802.1)
2.6
37.5:1
Applications
Polyacrylamide gel electrophoresis (PAGE) is ud for both high-resolution nucleic acid gels (e.g. quencing gels) as well as for almost all protein gels. Nucleic acid is, as a rule, parated in a TBE-buffer system, whereas proteins are mixed with SDS for a uniform negative load and parated with Tris/Glycine buffer (SDS-PAGE). A detailed description can be found Sambrook and Rusl’s Molecular Cloning 3rd Edition , CSHL Press New York, 2004 (Art. No. Y398.1) or in Proteins: Standard Methods of Molecular and Cell Biology , Eckert and Kartenbeck, Springer Verlag Heidelberg, 1997 (Art. No. L937.1).
Following tables provide a reference for typical gel mixes in SDS-gel electrophoresis (A) and the paration of nucleic acids (B).
A) SDS-PAGE
Separation range of SDS-gels Acrylamide concentration (%) 6 8 10 12 15 Separation range (kD)
50-200
30-95
20-80
12-60
10-43
Resolving gel (data apply to 20 ml gel solution) Gel concentration 6 % 8 % 10 % 12 % 15 % Aqua dest. (ml)
后会无期台词10.6 9.3 7.9 6.6 4.6 30 % acrylamide mix (ml) 4 5.3 6.7 8 10 30 % acryl- amide mix Tris (1.5 M, pH 8.8) (ml) 5 5 5 5 5 Gel concentration 6 % 8 % 10 % 12 % 15 % Aqua dest. (ml)
11.6 10.6 9.6 8.6 7.1 40 % acrylamide mix (ml) 3 4 5 6 7.5 40 % acryl- amide mix
in other wordsTris (1.5 M, pH 8.8) (ml)
5
5
5
5
5
Add in this order:
200 µl 10 % SDS solution (mix carefully, avoid bubbles) 200 µl 10 % ammonia persulphate solution (prepare freshly) 20 µl TEMED (mix carefully, avoid bubbles)
Pour gel immediately and overlay with isopropanol
Stacking gel (data apply to 5% gels) Gel volume 1 ml 3 ml 5 ml 8 ml 10 ml Aqua dest. (ml)
0.68 2.1 3.4 5.5 6.8 30 % acrylamide mix (ml) 0.17 0.5 0.83 1.3 1.7 Tris (1.0 M, pH 6.8) (ml) 0.13 0.38 0.63 1 1.25 SDS (10 % solution) (µl) 10 30 50 80 100 APS (10 % solution*) (µl) 10
30 50 80 100 30 % acryl- amide mix
TEMED (µl)
1 3 5 8 10 Gel volume 1 ml 3 ml 5 ml 8 ml 10 ml Aqua dest. (ml)conductivity
0.725 2.185 3.645 5.84 6.3 40 % acrylamide mix (ml) 0.125 0.375 0.625 1 1.25 Tris (1.0 M, pH 6.8) (ml) 0.13 0.38 0.63 1 1.25 SDS (10 % solution) (µl) 10 30 50 80 100 APS (10 % solution*) (µl) 10 30 50 80 100 40 % acryl- amide mix
TEMED (µl)
1
3
5
8
全国两会常用词汇10
* prepare freshly!
Be careful to mix the solution thoroughly before and after addition of SDS and TEMED. Avoid bubbles. Pour the stacking gel immediately and inrt the comb carefully.
B) Separation of nucleic acids
Denaturing TBE gels for paration of single stranded nucleic acids (e.g. quencing gels) (data apply to 100 ml gel solution) Gel concentration
local
内双化妆4 % 6 % 8 % Sequencing gel diluent (ml)*
复旦大学mba
74 66 58 25 % quencing gel concentrate with urea 25 % quencing gel concentrate (ml) 16 24 32 Gel concentration
4 % 6 % 8 % Aqua dest. (ad 90 ml) (ml)* app. 52 app. 4
5 app. 39 30 % acrylamide mix (ml) 13.3 20 26.5 30 %
acrylamide mix (29:1)
Urea (g)**
42 42 42 Gel concentration 4 % 6 % 8 % Aqua dest. (ad 90 ml) (ml)* app. 55 app. 50 app. 45 40 % acrylamide mix (ml)
10 15 20 40 % acrylamide mix (19:1 or 29:1) Urea (g)**federal rerve
42
42
42
*If required for resolution of condary structures the gel may be supplemented with formaldehyde by replacing 25 ml aqua dest. with 25 ml formaldehyde. **Results in gels with 42 % urea (7 M)
Add in this order:
10 ml 10 x TBE buffer*** (mix carefully, avoid bubbles, degas if required) 400 µl 10 % ammonia persulphate solution (prepare freshly) 50 µl TEMED (mix carefully, avoid bubbles)
Pour gel immediately and inrt the comb carefully
***Results in 45 % urea if quencing gel concentrate and quencing gel diluent are ud. If 50% urea are required replace 10 x TBE by the ready-to-u quencing gel buffer concentrate with 50 % urea (Ord. No. 3050.1).
TBE gels for electrophoresis of ds nucleic acid (data apply to 100 ml gel solution) Gel concentration 6 % 10 % 15 % Aqua dest. (ml)
69 56 39 30 %
acrylamide mix (29:1) 30 % acrylamide mix (ml) 20 33 50 Gel concentration 6 % 10 % 15 % Aqua dest. (ml)
74 64 51.5 40 %
acrylamide
mix (19:1 or 29:1)
40 % acrylamide mix (ml)
15
25
37.5
Add in this order:
10 ml 10 x TBE buffer (mix carefully, avoid bubbles, degas if required) 1 ml 10 % ammonia persulphate solution (prepare freshly) 60 µl TEMED (mix carefully, avoid bubbles)
Pour gel immediately and inrt the comb carefully
Variable Regulation of Pore Sizes Using Rotiphore® Gel A and B
The pore size of acrylic amide gels can be varied by regulating the total gel concentration (% T) and the percentage of the crosslink (% C). Gels with every desired T/C ratio can be produced with Rotiphore® Gel A and B:
V t= Total volume of gel casting solution (ml)
T = Gel concentration in % = % Acrylamide + % Bisacrylamide
C = % Crosslinking = (% Bisacrylamide x 100) / T
V a = Volume Gel A in ml V b = Volume Gel B in ml
Applying:
V a = (T x (100-C) x V t) / 3000 V b = (T x C x V t) / 200
Example: To prepare 100 ml gel solution with 10 % T and 2.7 % C, calculate as follows: V a = (10 x (100-2.7) x 100) / 3000 = 32.43 ml Gel A
V b = (10 x 2.7 x 100) / 200 = 13.5 ml Gel B
Combine 32.43 ml Gel A and 13.5 ml Gel B and fill up the volume to 100 ml with the usually ud buffer. Degas and add APS and TEMED, mix thoroughly while avoiding bubbles and pour the gel.
英语诗
Additional Reagents and Solutions:
Product Ord. No.
TEMED 2367
APS 9592
Acrylamide p.a., 4 x crist. 7906
Bisacrylamide 7867
Tris p.a. 4855
Glycine p.a. 3908
SDS ultra pure 2326
Urea X999
Rotiphore® NF Urea, fluorescence free A120
Rotiphore® Sequencing Gel Diluent 3047
Rotiphore® Sequencing Gel Buffer Concentrate 3050
Rotiphore® NF 10 x TBE Buffer, fluorescence free A118
Rotiphore® 10 x TBE Puffer 3061
Rotiphore® 10 x SDS PAGE Buffer 3060
Roti®-Stock 20 % SDS Fertiglösung 1057
For further data, safety information, or package sizes plea e our catalogue or online at
