This week we will run the PCR reactions on an agarose gel and analyze the result

Question

This week we will run the PCR reactions on an agarose gel and analyze the results. Safety notes: Ethidium bromide is a potent mutagen. Wear gloves when handling containers containing ethidium bromide, when handling gels that have been stained in ethidium bromide, and when working with the computer attached to the gel-doc system. Protocol I. Pouring agarose gels I. Using 10x TAE, prepare 50 ml of 3% (w/v) agarose in 1x TAE. g of agarose ml of 10x TA ml of DI water Perform the necessary calculations as part of the pre-lab assignment. During lab when you get your pre-lab handed back, if your calculations were correct, record them in your lab notebook and proceed with making your 3% agarose in 1x TAE. If you need to redo your calculations, check them with Rachel before proceeding 2. The process of preparing the gel mold, heating the agarose, and pouring the gel will be demonstrated. Once you have your gel poured, wait until it is fully cooled before proceeding. Il. Loading and running the gel 1. Plan out and record which PCR reactions will go into which wells 2. Using a fresh tip each time, add 10 1 of Orange G loading dye to each sample and mix well 3. Once you have your samples ready to load, it will be demonstrated how to set up the electrophoresis chamber and how to load your samples. You will load 20 of each PCR rxn and 10 1 of a DNA ladder (a sample containing known sizes of DNA fragments). 4. Run the gel at 100 V for 30 minutes. Be sure to stop the gel before the orange dye runs off the end of the gel. The orange dye co-migrates with DNA that is 50 bp in length 5. Stain the gel in ethidium bromide on the shaker table for 30 minutes. 6. Take a photo of your gel using the gel-doc system. This process will be demonstrated for you. IlII. Analysis 1. Determine the approximate molecular weight of each band on you gel based on the knowrn

Explanation / Answer

Preparation of 50 mL 3% w/v agarose gel in 1X TAE:

i. 3% agarose = 3 g in 100 mL

= 1.5 g in 50 mL

ii. For 50 mL of 1 X TAE, take 5 mL 10 X TAE and add 45 mL of water

= 5 mL 10X TAE + 45 mL water

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