215-Lab9-Agarose_gel

215-Lab9-Agarose_gel - Lab 9 Preparing and running an...

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Lab 9 –Preparing and running an Agarose Gel 9-1 Lab 9: Preparing and running an Agarose Gel A) Prepare the agarose gel . (Note: It is helpful to view the online video of making and loading a gel that is posted in the Animations folder) 1. Melt 80 ml of 1% agarose gel in 1X TAE buffer in the microwave. a) Add the 0.8 g of agarose to 40 ml of 1X TAE stock in a 250 ml bottle. b) Swirl to briefly suspend the agarose and make sure it is dispersed. c) Heat the mixture for 2 min at full power. Make sure the cap is loose so that pressure will not build in the bottle when it is heated. Wearing an oven mitt, periodically take out the bottle and swirl it to mix any agarose that has not melted. Be Careful! — The bottle is very hot and may boil over when mixed. Never hold the bottle from above, since the rising steam may burn your hand. While swirling the bottle, look for undissolved agarose that appears as clear specks in the solution. If necessary heat the mixture again to completely dissolve the agarose. d) Add 40 ml of of 1X TAE stock to the melted agarose. This will help the agarose cool faster. e) Add 5 μ l of 10 mg/ml Ethidium bromide (EtBr) to the bottle and swirl REMEMBER, EtBr IS A POTENTIAL MUTAGEN. ALWAYS WEAR GLOVES WHEN WORKING WITH EtBr. IF YOU HAVE ANY QUESTIONS OR CONCERNS, TALK TO ONE OF THE INSTRUCTORS. f) Let the agarose slowly cool until it is not to hot to hold in hand (approx. 60 o C) This should take about 10 minutes. During this 10 minute cooling period you should periodically swirl the bottle gently to prevent any lumps from forming due to uneven cooling. 3. Pour the cooled agarose into the gel casting box with one 14-well comb in place and let it harden for approximately 30 minutes. Before adding the agarose, make sure the gel apparatus is clean, dry, and on a level part of
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215-Lab9-Agarose_gel - Lab 9 Preparing and running an...

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