07 - Lion Family Reunion: Conservation Biology Genetics
Agarose Gel Electrophoresis Conduct agarose gel electrophoresis to determine the lions’ restriction fragment length polymorphisms (RFLP). This will be used to determine the lions’ haplotypes for reintroduction to the wild.
Concentrated buffer
Distilled water
Agarose
60°C
Flask
Caution! Flask will be HOT!
WAIT
POUR
60°C
ADD SYBR Safe
60°C
1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A). 2. MIX agarose powder with 1X buffer in a 250 mL flask (see Table A). 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water). 4. COOL agarose to 60°C with careful swirling to promote even dissipation of heat. 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch. 6. Before casting the gel, ADD diluted SYBR ® Safe to the molten agarose and swirl to mix (see Table A). 7. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies. 8. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells.
Individual 0.8% UltraSpec-Agarose™ with SYBR ® Stain
7 x 7 cm
0.6 mL
29.4 mL
0.23 g
30 mL
30 µL
10 x 7 cm*
1.0 mL
49.0 mL
0.39 g
50 mL
50 µL
14 x 7 cm
1.2 mL
58.8 mL
0.46 g
60 mL
60 µL
* Recommended gel volume for the EDGE™ Integrated Electrophoresis System.
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