Bio-Rad Criterion™ Blotter - Manual (Page 11)

10. These variables will change total resistance and thus the current readings:
• Alterations in buffer make-up, i.e., addition of SDS, or changes in ion concentration
due to addition of acid or base to adjust the pH of the buffers.
• Gel pH, ionic strength, and percentage of acrylamide, especially if the gel has not
been properly equilibrated.
• Number of gels; current increases slightly as the number of gels increases.
• Transfer temperature; current increases when temperature increases.
• Time in transfer at which reading was taken; current normally increases as the
buffering capacity diminishes with progress of the run.
3.3 Buffer Formulation
All formulas provided below are for a total volume of 1 liter of buffer. 1.3 liters of buffer
are required for the Criterion Blotter cell plus another 300 to 500 ml for assembly procedure
for a total of 1.6 to 1.8 liters of buffer. It is efficient to use premixed buffer concentrates from
Bio-Rad where available.
Do not add acid or base to adjust pH of the following buffers*. Methanol should be
analytical reagent grade, because metallic contaminants in low grade methanol will plate on
the electrodes. Always add methanol/ethanol last.
Note: Some pH electrodes will not perform a proper measurement for the pH of Tris buffers.
If the pH of the buffer is off, check to make sure the electrode is designed to work with Tris
buffers. If the pH electrode functions properly for Tris buffers and the pH is below 8.0, remake
the buffer.
1. SDS PAGE gels
Towbin Buffer with 20% Methanol
25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3
a) Use Premixed buffer concentrate solution:
100 ml of 10X Tris/Glycine buffer (catalog number 161-0734 1L bottles or catalog
number 161-0757 5L cube)
700 ml of dd H
2
O
200 ml of methanol
b) From dry reagents:
Mix 3.03 g Tris, 14.4 g glycine in 600 ml of dd H
2
O, add distilled deionized water
(dd H
2
O) to 800 ml, add 200 ml Methanol.
Towbin Buffer with 10 % Methanol
25 mM Tris, 192 mM glycine, 10% v/v methanol, pH 8.3
a) Use Premixed buffer concentrate solution:
100 ml of 10X Tris/Glycine buffer (catalog number 161-0734 1L bottles or catalog
number 161-0757 5L cube)
800 ml of dd H
2
O
100 ml of methanol
b) From dry reagents:
Mix 3.03 g Tris, 14.4 g glycine in 600 ml of dd H
2
O, add distilled deionized water
(dd H
2
O) to 900 ml, add 100 ml Methanol.
* pH can be adjusted for CAPS buffer
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