Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 24

Materials Electrophoresis Buffer concentrate, TAE (40X or 50X) or LB buffer (10X or 20X) Beakers, 600 mL Agarose Balance, tabletop milligram Weigh boat, 3.5”×3.5” Lab scoops Media bottle, 250 mL Permanent lab marker pens Microwave oven Hot hands protector Glasses, safety Gel box, horizontal, for agarose gels Beakers, 50 mL Water bath, 65°C Procedure • A 0.8% gel is prepared in the following procedures since that is the appropriate concentration for resolving a variety of DNA fragment lengths. • Prior to step 1, prepare 500 mL of 1X electrophoresis buffer from the buffer concentrate provided. The 1X buffer will be used in this and in the following activity. Use the C1 V1 = C2 V2 equation. Record the calculations and recipe in your notebook. 1. Make 100 mL of 0.8% agarose in 1X electrophoresis buffer solution. Determine the recipe for a 0.8% gel, below. 0.8% of 100 mL = _________ g of agarose in a total volume of 100 mL of 1X electrophoresis buffer 2. Obtain a clean 250-mL media bottle and cap. Label it. 3. Weigh out the required mass of powdered agarose in a weigh boat. Add it to the media bottle. 4. Measure out enough 1X electrophoresis buffer to prepare a total of 100 mL of agarose and buffer mixed together. Add the buffer slowly to the agarose in the media bottle, swirling it to mix. 5. Loosely cap the top of the media bottle. Swirl the flask gently to suspend the agarose in the buffer. 6. To dissolve the agarose in the buffer, microwave the suspension for 4 minutes at 50% power. 7. Wearing bottle holders and safety goggles, lift the beaker toward the light (but away from your face), and very slowly and gently swirl it. Caution: Do not swirl too fast because it could boil over. Look to see if all the agarose crystals have dissolved. Agarose crystals that do not dissolve will impede molecular motion through the gel and will affect electrophoresis results. If any agarose crystals are still floating in the buffer, reheat the solution at 50% for 2 minutes more. 8. Using hot hand protectors, place the hot dissolved agarose solution on a fireproof lab tabletop. Figure 4.18.  If the agarose solution is not to be used 9. The solution must cool to immediately, it can be kept hot in a 65°C water bath until it is approximately 65°C before time to cast the gels. pouring it (see Figure 4.18). Photo by author. 10. Prepare the gel tray found in your gel box for pouring (casting) a 7mm thick agarose gel. Note: Different gel boxes have different types of gel-casting trays. Some trays have gates at the ends that are screwed into an upright position for gel casting. Some gel trays use tape to seal the ends. Some gel trays have rubber gaskets that seal the ends of the tray in the gel box for pouring. Your instructor will show you how your gel tray should be set up for gel pouring. 11. When the agarose solution is about 65°C (just barely cool enough to hold), pour about 30 mL of it into the gel tray. The gel should be about 7mm thick. 12. Quickly place a six-well comb into the notches at the end of the gel tray. Make sure the comb DNA Isolation and Analysis 89