Required Reagents & Recipes; Electrophoresis Buffers - VWR Peqlab PerfectBlue Mini S Instruction Manual

Instruction Manual PerfectBlue
REQUIRED REAGENTS & RECIPES

Electrophoresis buffers

Electrophoresis buffers supply the ions necessary for electrophoresis and establishing a certain pH value
in which the target molecule adapts to the required electric charge. Nucleic acids will be negatively
charged in an alkaline to neutral surrounding. Additionally, electrophoresis buffers often contain rea-
gents which protect the target molecule from degradation (e.g. EDTA, which complexes bivalent cations
and therefore inhibits DNases). If electrophoresis under denaturing conditions is desired (like for the
electrophoresis of RNA), electrophoresis buffers will additionally contain reagents that eliminate the
formation of secondary structures. You will find recipes below for TAE and TBE, two of the most com-
monly used buffers for the electrophoresis of DNA. If the intention is to eventually isolate DNA from the
gel, TAE buffer should be chosen. In comparison to TBE, migration will be faster and a better resolution
of supercoiled DNA will be achieved when using TAE. However, because of TAE's limited buffering
capacity, TBE should be selected for performing extended electrophoresis separations and if the electro-
phoresis chamber does not possess a system for buffer recirculation. VWR's PerfectBlue 'Revolution'
Systems are equipped with an internal buffer recirculation system that prevents the formation of pH and
ion gradients during extended runs. Since agarose tends to create finer pore sizes and a more solid
matrix in TBE, diffusion of DNA will be reduced and a more discrete band pattern will be achieved.
TAE (Tris-Acetate-EDTA) Buffer
1 x working solution:
50 x stock solution (1 L):
TBE (Tris-Borate-EDTA) Buffer
0.5 x working solution*:
5 x stock solution (1 L)**:
* 0.5 x TBE is sufficient for agarose gel electrophoresis. For vertical electrophoresis in polyacrylamide gels,
1 x TBE is often applied due to the comparatively smaller buffer reservoirs of vertical electrophoresis chambers.
** 5 x TBE stock solutions tend to precipitate during long storage periods and should get remade. Because of this
property, higher concentrations of TBE stock solutions should be avoided.
VWR_v0617_E
Horizontal Mini Gel Systems
TM
40 mM Tris-acetate, 1 mM EDTA
242 g Tris-Base
57.1 ml Glacial acetic acid
100 ml 0.5 M EDTA (pH 8.0)
Adjust volume to 1L using distilled H
45 mM Tris-Borate, 1 mM EDTA
54 g Tris-Base
27.5 g Boric acid
20 ml 0.5 M EDTA (pH 8.0)
Adjust volume to 1L using distilled H
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