Team:WashU/Protocols/SDSPAGE
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<!--Thanks to Cambridge iGEM '11 for the above instructions. The original source can be found here: https://2011.igem.org/Team:Cambridge/Protocols/Gel_Electrophoresis_of_Protein#/Protocols/Gel_Electrophoresis_of_Protein--> | <!--Thanks to Cambridge iGEM '11 for the above instructions. The original source can be found here: https://2011.igem.org/Team:Cambridge/Protocols/Gel_Electrophoresis_of_Protein#/Protocols/Gel_Electrophoresis_of_Protein--> | ||
<!--NOTE: We have modified the protocol to suit our purposes by using a different protocol found on the internet. Thus, the recipes for making the separating and stacking gels are different from what was on the Cambridge website.--> | <!--NOTE: We have modified the protocol to suit our purposes by using a different protocol found on the internet. Thus, the recipes for making the separating and stacking gels are different from what was on the Cambridge website.--> | ||
- | {{ | + | {{WashUbackprotocols}} |
- | + | <div id = "aprotocol"> | |
==SDS-PAGE Gel Electrophoresis== | ==SDS-PAGE Gel Electrophoresis== | ||
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|- | |- | ||
|dH<sub>2</sub>O | |dH<sub>2</sub>O | ||
- | |3.075 | + | |3.075 ml |
|- | |- | ||
|30% acrylamide/0.8% methylene bisacrylamide | |30% acrylamide/0.8% methylene bisacrylamide | ||
- | |0.67 | + | |0.67 ml |
|- | |- | ||
|4x Upper Tris | |4x Upper Tris | ||
- | |1.25 | + | |1.25 ml |
+ | |- | ||
+ | |10% APS | ||
+ | |.005 ml | ||
+ | |- | ||
+ | |20% SDS | ||
+ | |.025 ml | ||
+ | |- | ||
+ | |TEMED | ||
+ | |.005 ml | ||
|- | |- | ||
- | |||
- | |||
|} | |} | ||
</center> | </center> | ||
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:<li>Gently mix the stacking gel solution. Excess Aeration will interfere with the polymerization of the gel.</li> | :<li>Gently mix the stacking gel solution. Excess Aeration will interfere with the polymerization of the gel.</li> | ||
:<li>Get ready a Pasteur pipette with a bulb</li> | :<li>Get ready a Pasteur pipette with a bulb</li> | ||
- | :<li>Pour the ethanol off the separating gel. Rinse the gel assembly out with several changes of dH<sub>2</sub>O | + | :<li>Pour the ethanol off the separating gel. Rinse the gel assembly out with several changes of dH<sub>2</sub>O. Mix well. Use a Pasteur pipette to fill the glass plates up to the top with stacking gel solution. </li> |
- | + | ||
:<li>Carefully insert the comb. Be sure that no air bubbles are trapped to the ends of the teeth. </li> | :<li>Carefully insert the comb. Be sure that no air bubbles are trapped to the ends of the teeth. </li> | ||
:<li>Let the stacking gel polymerize for 30 minutes. </li> </ol> | :<li>Let the stacking gel polymerize for 30 minutes. </li> </ol> | ||
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:#Check the volume of the wells in the gel. Mix protein samples in volume ratio of 3:1 with 4x Sample Buffer in a microcentrifuge tube including the protein marker. | :#Check the volume of the wells in the gel. Mix protein samples in volume ratio of 3:1 with 4x Sample Buffer in a microcentrifuge tube including the protein marker. | ||
:#*'''''Note:''''' ''If you don't have enough samples to completely fill the number of wells dilute the 4x Sample Buffer with D.I water and load this. | :#*'''''Note:''''' ''If you don't have enough samples to completely fill the number of wells dilute the 4x Sample Buffer with D.I water and load this. | ||
- | :#Submerge the protein in boiling water bath (100<sup>o</sup>c works for me) for 2 mins. | + | :#Submerge the protein in boiling water bath (100<sup>o</sup>c works for me) for 2 mins. Alternatively, you could use a 37<sup>o</sup>C water bath for 30 minutes. |
:#Centrifuge for 20 secs at 12,000 rpm. You are now ready to load your samples into the gel. | :#Centrifuge for 20 secs at 12,000 rpm. You are now ready to load your samples into the gel. | ||
Latest revision as of 03:42, 4 October 2012