Lab Protocols

Materials: -All materials are EtBr exposed, so wear gloves! -Materials are kept on a tray on the lab bench 1. TAE solution 2. Agarose 3. EtBr 4. Plate set-up 5. DNA ladder (in fridge) 6. PCR product For 70mls of 1% gel solution: 1. Add 70 mls of 1X TAE to the flask 2. Add 0.7g of agarose to the flask 3. Microwave the solution for 1:30 mins, watching for bubbles 4. Microwave again until the solution clears and bubbles but does not overflow 5. Allow the solution to cool to 50 degrees celsius (warm to touch) 5. Under the hood, add 3.5uL of EtBr to the solution (before it cools completely!) 5. Stir the solution gently so that the EtBr dissolves 6. Once dissolved, pour the solution onto the gel plate set-up. Make sure the comb is properly oriented and that the screws are tightened. 7. Allow plate to cool for at least 25 mins 8. Place plate into buffer (TAE) and gently remove the comb 9. Add enough TAE to cover the wells 10. Use a DNA ladder, 20 uL in one well 11. Sp...

 I. BACKGROUND & PURPOSE Gel electrophoresis is a useful method to separate and/or identify proteins and nucleic acids. In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins are separated largely on the basis of polypeptide length, and so their molecular weight can also be estimated. SDS does however denature the protein, so activity stains cannot be used to identify particular enzymes. Described below is the protocol for preparing and using Laemmli discontinuous gels. In this system, two sequential gels are actually used; the top gel, called the stacking gel, is slightly acidic (pH 6.8) and has a low (5.5%) acrylamide concentration to make a porous gel. Under these conditions proteins separate poorly but form thin, sharply defined bands. The lower gel, called the separating, or resolving gel, is more basic (pH 8.8), and has a higher polyacrylamide content (in our case, 12%), which causes the gel to have narrower channels or pores. As a protein, concen...

A Protein Electrophoresis Protocol Hahn Lab, 2001 1. After electrophoresis of protein gel, transfer gel to round staining tray. Add ~200 ml protein gel stain. 2. Microwave for ~45 sec until the solution just starts to boil. Incubate at room temp with gentle shaking for 10-15 min. Heating allows the gel to stain faster. Alternatively, soak gel instain for 1 hr at room temperature. 3. Pour stain back into bottle for later reuse. Rinse gel with water. Add ~200 ml protein gel destain. Microwave ~45 sec until the solution just starts to boil. Incubate with gentle shaking at room temp for ~15 min. Alternatively, soak gel with gentle shaking at room temp ~45 min. 4. Discard destain and add remainder of stain. Microwave as before and incubate with shaking at room temp until gel is destained the desired amount. Alternatively, the microwave step can be omitted and the gel destained an additional hour or overnight. Solutions: Protein gel stain Add to a 500 ml bottle: 1.2 g...

Materials * Polyacrylamide pre-cast gel * SimplyBlue™ Coomassie protein stain, 1x pre-mixed solution (Coomassie blue dye binds to proteins (arginine, the aromatic amino acids, and histidine) allowing protein bands to be visualized in polyacrylamide gels.) * Plastic container * Gel separator Procedure 1. After electrophoresis, remove the gel from the gel box and plates using a gel separator 2. Place it in a clean plastic container and cover with ultrapure water 3. Microwave the gel on high for 1 minute until the solution almost boils (do not overheat) 4. Shake the gel on a shaker for 2 minute and discard the water 5. Repeat steps 3 and 4 two more times 6. After the last wash, add 30 ml of SimplyBlue™ Coomassie protein stain and microwave on high for 45 seconds to 1 minute until the solution almost boils 7. Shake the gel on a shaker for 10 minutes 8. Wash the gel in 100 ml of ultrapure water for 10 minutes on a shaker 9. Add 20 ...