<noautolink> ---+ 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 %BLUE%denaturing%ENDCOLOR% 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. For %GREEN%nondenaturing%ENDCOLOR% gels, use the [[http://www.nationaldiagnostics.com/product_info.php/products_id/37][AccuGel system]]. The concentrated stock solution is 40%. Dilute the stock to get the desired gel percentage in your final volume. Add 1/10th volume 10x TBE buffer, then ddH<sub>2</sub>O for the rest of the volume. %TABLE{dataalign="center"}% | *Spacer Width* | *Gel Height* | *Gel Width* | *Gel Vol* | *TEMED Vol* | *10% APS Vol* | | 1 mm | 14.5 cm | 16.5 cm | 25 ml | 25 µl | 200 µl | | 1 mm | 28 cm | 16.5 cm | 50 ml | 50 µl | 400 µl | | 1.5 mm | 14.5 cm | 16.5 cm | 30 ml | 30 µl | 240 µl | | 1.5 mm | 28 cm | 16.5 cm | 75 ml | 75 µl | 600 µl | Add 10 µl of TEMED per 10 ml volume, then 50 µl of 10% w/v APS per 10 ml volume to initiate polymerization of the gel. Pour it quickly into the glass plates with spacers clamped between them. TEMED is N,N,N',N'-tetramethylethylenediamine. APS is ammonium persulfate. Together, they create the radicals to polymerize the gel. TEMED and 10% APS should be stored at 4°C. APS solutions should be freshly prepared for best results. For gels that are 1 mm or less in thickness, you can prop the glass plates horizontally and pour the gel without using a casting boot. Surface tension will keep the gel solution from coming out the bottom. %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 For %BLUE%denaturing%ENDCOLOR% gels, use the 2x formamide sample loading buffer. For %GREEN%nondenaturing%ENDCOLOR% gels, use the 5x glycerol sample loading buffer. Be sure to wash buffer out of the wells using a syringe immediately 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 You will need a high voltage power supply to run the large vertical polyacrylamide gels. Generally denaturing gels are run at a constant electrical power (Watts). This maintains a certain heated gel temperature during the run. *For the 16.5 cm x 28.5 cm gels, use 35 W.* Using a higher voltage can cause excessive heating that will crack the glass plates. ---++ Reagents All reagents should be prepared with RNAse, DNase free water. ---+++ 2x Formamide Loading Buffer | 32 ml | Deionized formamide | | 8 ml | 10x TBE Buffer | | 16 mg | Bromophenol Blue | Makes 40 ml. Use for %BLUE%denaturing%ENDCOLOR% gels. Note: Many recipes for this do not add the TBE buffer. This is fine if you are loading straight from a reaction which has buffer in it, but may cause problems if your sample is in pure H<sub>2</sub>O (for example, after EtOH precipitation). The final concentration of EDTA in this buffer at 1x is 10 mM. ---+++ 5x Glycerol Loading Buffer | 20 ml | 10x TBE Buffer | | 20 ml | Glycerol | | 16 mg | Bromophenol Blue | Makes 40 ml. Use for %GREEN%nondenaturing%ENDCOLOR% gels. Note: Many recipes for this do not add the TBE buffer or substitute sucrose for glycerol. ---+++ 6x Urea Loading Buffer | 24 g | Urea | | 2 ml | 0.5 M EDTA | | 100 µl | 1 M Tris pH 7.5 | | 16 mg | Bromophenol Blue | Add ddH<sub>2</sub>O to 50 ml. Use for Use for %BLUE%denaturing%ENDCOLOR% gels. Note: Bromophenol Blue is optional. ---+++ 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: r8 - 2013-06-07 - AlvaroRodriguez