Although you can use polyacrylamide gels for separation of DNA, they are better suited for protein separation and characterization. They are widely used to study molecular weight, charge, structure and purity of proteins. The electrophoretic mobility of proteins enables them travel through the network of acrylamide polymers when current is applied. Proteins separate according to their size.
Things You'll Need
- Glass plates
- Safety gloves
- 150 mL flask
- 26.6 mL of 29% Acrylamide plus 1% N,N-Methylenebisacrylamide
- 52.7 mL of water
- 20 mL of 5X Tris/BorateEDTA (TBE) Buffer
- 0.7 mL of 10% Ammonium Persulfate
- 35 micro liters of TEMED
Put on gloves. Thoroughly clean and dry the glass plates, comb and spacers.
Put the larger plate on a clean dry surface. Place spacers on both sides and cover with smaller plate. Carefully align the plates with the spacers on two sides and the bottom. Clamp the three sides of the plates to seal and hold them together.
Calculate the amount of gel volume required for the size of plates and thickness of spacers.
Determine percent of polyacrylamide solution needed for your experiment. For 100 mL of a 8% polyacrylamide gel, mix 26.6 mL of 29% Acrylamide plus 1% N,N-Methylenebisacrylamide, 52.7 mL of water, 20 mL of 5X TBE and 0.7 mL of 10% Ammonium Persulfate in a small flask. Add 35 micro liters of TEMED to the solution and mix gently. Work quickly after adding TEMED. Fill a syringe with the solution and carefully dispense contents in empty space between plates until solution is slightly below smaller plate.
Carefully insert the well-forming comb between the large and small plates on the top of the assembled plates. Let the gel polymerize completely for about an hour.
Gently remove the comb. Flush the wells with 1X TBE to clean them. Take off the clamps and place the gel in the electrophoresis apparatus according to manufacturer's directions. The polyacrylamide gel is ready to use for your experiment.
Tips & Warnings
- Clean the glass plates thoroughly before pouring the gel. Any smudges or oil on the plated will result in air bubbles and hinder sample migration.
- Because acrylamide is toxic, take upmost care when you work with it. Always wear gloves when handling acrylamide in any form. Use an automatic pipette to measure acrylamide solution and put on a face mask when using acrylamide powder.
How Long Does Meat Stay Good in the Refrigerator?
Eating food that's left in the refrigerator too long can cause serious and violent illnesses. The following guidelines are issued by the...
How to Make Gel Candles
Gel candles, also known as jelly candles, are gaining in popularity because they burn longer than wax candles and are easy to...
How to Make Saline Solution to Clean a Wound
If you are out camping or hiking, and one of your companions trips and suffers a wound, you will have to help...
How to Make Your Own Light Saber Toy
Make your own Sith inspired double-blade cross guard lightsaber from the new Star Wars movie with items found at the hardware store.
How to Make Your Money Work for You
For many people, the importance of working for money is driven home at an early age, which is why you see kids...
How to Calculate PH of Buffer Solutions
A buffer is an aqueous solution designed to maintain a constant pH, even when exposed to small amounts of acids or bases....
How to Calculate the Percent Weight Per Volume
Percent weight per volume is defined as the grams of solute in 100 milliliters of solution. The calculation provides information about the...
How to Make Cosmetic Gel
At the most basic level, gels are active ingredients suspended in a base of water and a thickening agent, such as xanthan...
The Ingredients in Ultrasound Gel: Polyacrylamide Gel's Properties
Polyacrylamide is a gel created by the process of electrophoresis, a technique used in biochemistry and molecular biology to separate macromolecules. It...