---+ Polyacrylamide Gel Electrophoresis Our gel rigs and supplies are from [[http://www.cbsscientific.com/][CBS Scientific]]. The [[http://www.nationaldiagnostics.com/articles.php/tPath/1_3][National Diagnostics Website]] has very helpful background on RNA/DNA polyacrylamide gels. ---++ Pouring the Gel For denaturing urea gels, we use the [[http://www.nationaldiagnostics.com/images/ec833_protocol.pdf][SequaGel system]]. Check out the link to determine how to mix up a gel of the proper percentage. %TABLE{dataalign="center"}% | *Spacer Width* | *Gel Height* | *Gel Width* | *Gel Vol* | *TEMED Vol* | *10% APS Vol* | | 1 mm | 28 cm | 16.5 cm | 50 ml | 20 µl | 400 µl | | 1 mm | 14.5 cm | 16.5 cm | 25 ml | 10 µl | 200 µl | TEMED is N,N,N',N'-tetramethylethylenediamine. APS is ammonium persulfate. Together, they create the radicals to initiate polymerization of the gel. TEMED and 10% APS should be stored at 4°C. APS solutions should be freshly prepared for best results. %RED%Acrylamide is a neurotoxin before it is polymerized.%ENDCOLOR% You are working with it in a liquid solution where spills may happen. Wear gloves, a lab coat, and safety glasses when working with polyacrylamide gels. ---++ Loading the Gel Formamide sample loading buffer. Be sure to wash urea out of the wells using a syringe before loading the gel. %TABLE{dataalign="center"}% | *Spacer Width* | *Gel Width* | *Well Height* | *Well Width* | *# Wells in Comb* | *Max Sample Vol* | | 1 mm | 16.5 cm | 15 mm | 8 mm | 12 | 120 µl | | 1 mm | 16.5 cm | 15 mm | 4 mm | 20 | 60 µl | | 1 mm | 16.5 cm | 15 mm| 2 mm | 30 | 30 µl | For sharp bands, you should load to much less than the maximum well volume. ---++ Running the Gel Generally denaturing polyacrylamide gels are run at a constant electrical power (Watts). This maintains a certain heated gel temperature during the run. For the 16.5 cm width gels, use 25 W. ---++ Reagents All reagents should be prepared with RNAse, DNase free water. ---+++ 2x Loading Buffer | 36 ml | Deionized formamide | | 4 ml | 10x TBE Buffer | | 16 mg | Bromophenol Blue | Makes 40 ml. The final concentration of EDTA in this buffer at 1x is 10 mM. ---+++ 10x TBE Buffer | 108 g | Tris base | | 55 g | Boric acid | | 40 ml | 0.5 M EDTA, pH 8.0 | Add ddH<sub>2</sub>O to 1 L. ---+++ 0.5 M EDTA, pH 8.0 1 Dissolve 186.1 g Na<sub>2</sub>EDTA in 700 ml ddH<sub>2</sub>O. 1 Adjust pH to 8.0 with 10 M NaOH. 1 Add ddH<sub>2</sub>O to 1 L.
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Topic revision: r3 - 2012-02-19 - JeffreyBarrick