Solution of Acrylamide and N, N^'-Methylenbisycrylamide 

Cat. No.	Composition Acrylamide:Bis	% C	% T	Applications
10679	19:1	5.0	40	DNA Sequencing
10680	29:1	3.3	40	IEF, SDS PAGE
10681	37.5:1	2.6	40	SDS PAGE
10687	29:1	3.3	30	IEF, SDS AGE
10688	37.5:1	2.6	30	SDS PAGE

 

The SERVA Acrylamide/Bis solutions are applicable to all PAGE methods. Acrylamide/Bis solution 29:1 is also suitable for preparation of a gel according to Schägger and Jagow ¹⁾. The amount of gel solutions (20 ml) is sufficient for 2 vertical slab mini gels (100 mm x 80 mm x 1,4 mm).

Product	For final concenration of gel (% T) Seperating gel (10 ml)								Stacking gel  (10 ml)
	7 %		10 %		12.5 %		15 %		5 %
	30 %*	40 %*	30 %	40 %	30 %	40 %	30 %	40 %	30 %	40 %
Acrylamide/Bis Solution %*	2.33 ml	1.75 ml	3.33 ml	2.50 ml	4.17 ml	3.13 ml	5.00 ml	3.75 ml	1.67 ml	1.25 ml
Seperating gel buffer 4 x	2.50 ml	2.50 ml	2.50 ml	2.50 ml	2.50 ml	2.50 ml	2.50 ml	2.50 ml	—	—
Stacking gel buffer 4 x	—	—	—	—	—	—	—	—	2.50 ml	2.50 ml
dist. H2O	5.07 ml	5.65 ml	4.03 ml	4.90 ml	3.29 ml	4.27 ml	2.46 ml	3.65 ml	5.79 ml	6.15 ml
SDS, 10 % Solution (Cat. No. 20763)	0.1 ml	0.1 ml	0.1 ml	0.1 ml	0.1 ml	0.1 ml	0.1 ml	0.1 ml	0.1 ml	0.1 ml
TEMED (Cat. No. 35925)	0.015 ml	0.015 ml	0.015 ml	0.015 ml	0.015 ml	0.015 ml	0.015 ml	0.015 ml	0.015 ml	0.015 ml
APS, 10 % Solution (Cat. No. 13375)	0.03 ml	0.03 ml	0.03 ml	0.03 ml	0.03 ml	0.03 ml	0.03 ml	0.03 ml	0.03 ml	0.03 ml

 

*The composition of the acrylamide/bis solution (acrylamide content: Crosslinker) depends on the application.
For standard SDS-PAGE, we recommend the Acrylamide/Bis Solution 29:1 (cat. no. 10680) or 37.5:1 (cat. no. 10681).

 

Required Buffers

Seperation gel buffer, 4x (1,5 M Tris/HCI pH 8.8)

• Dissolve 18.2 g Tris (cat. no. 37190) in water (bidest.)
• Adjust pH 8.8  (approx. 2.8 ml HCl, 32 %)

Stacking gel buffer, 4 x (0,5 M Tris/HCI pH 6.8)

• Dissolve 6.1 g Tris (cat. no. 37190) in water (bidest.) 
• Adjust pH 6.8  (approx.  4.4 ml HCl, 32 %)

 

Preparation

1. Mix acrylamide/bis solution, buffer and water in separate beakers.
2. Deaerate the solutions briefly (1 to 3 min ad vacuo).
3. Add to separating gel solution: 10 % SDS solution (w/v in water), TEMED and APS solution (w/v 10 % of ammonium persulfate), gently swirl to mix without incorporating air into the mixture.
4. Casting the separating gel: fill slab gel sandwich with separating gel solution up to approx. two thirds. The remaining volume for the stacking gel should refer to a separation distance of 1.5-fold of the depth of the wells of the comb to be used. Overlay with isopropanol immediately (0.1 ml), let polymerize (approx. 15 - 20 min).
5. Add 10 % SDS in water to the stacking gel solution, then TEMED and 10 % ammonium persulfate solution (APS), swirl gently to mix without incorporating air into the mixture.
6. Casting the stacking gel: fill up the slab gel sandwich to the top and let polymerize.

Literature:
1) Schägger H. & von Jagow, U. (1987) Anal. Biochem. 166, 368-379 2.
2) Laemmli, U.K. (1970) Nature 227, 680-685.