Bi1x_2009_W5 - Bi 1x Spring 2009 Week 5 Session 1 o Agarose...

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Page 1 of 5 Bi 1x, Spring 2009 Week 5 Session 1 o Agarose gel electrophoresis of PCR products Session 2 o TOPO cloning reaction o Bacterial transformation and plating Session 1 – Agarose gel preparation and electrophoresis In week 4, you loaded and ran agarose gels that your TAs had prepared for you. Today, you will run your pond sample PCR products on a gel that you prepare yourself. Note: Each small gel box can accommodate two individual’s samples. The large gel box holds up to eight individual’s samples. 1. Set up your gel box for casting: i. For the small boxes, rotate the gel tray inside the gel box so that the open, rubber gasketed sides are blocked by the sides of the box. Place a purple comb in, 1.5 mm side down. ii. For the large gel box, place the tray into the casting apparatus. Insert both combs, one at the top of the tray and one at the midway point. Ask a TA if you are unsure about the set up! 2. Prepare 50 ml (if you are using a small gel box) or 100 ml (if you are using the large gel box) solution of 2% agarose in TAE (tris acetate EDTA) buffer. For the 50 ml solution: Mix 1 gram of agarose with 50 ml TAE. For the 100 ml solution: Mix 2 grams of agarose with 100 ml TAE. TAE stands for Tris-acetate plus EDTA. Tris-acetate is a buffer that maintains proper pH control during gel electrophoresis. EDTA is a chelator of divalent ions. EDTA is often used in experiments involving DNA, since DNA-degrading enzymes (DNases) require the divalent ion Mg 2+ for activity. 3. To melt the agarose, heat the suspension in a microwave until it begins to boil. This may take less than a minute. Gently swirl the flask from time to time. Be extra cautious, since super heated solutions of agarose have a tendency to boil explosively. Wear protective mittens when you handle the hot flask and keep it away from your face!
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Page 2 of 5 Continue heating and swirling the agarose solution until it is completely
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Bi1x_2009_W5 - Bi 1x Spring 2009 Week 5 Session 1 o Agarose...

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