Neutral polyacrylamide gel electrophoresis
Neutral polyacrylamide gel electrophoresis
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Neutral polyacrylamide gel electrophoresis
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Acrylamide Methylenebisacrylamide Ammonium persulfate Ethanol Gel loading buffer KOH Methanol Silicification solution TBE Electrophoresis buffer TEMED
Clips or tin paper clips Electrophoresis apparatus Glass plates Combs and spacers Gel sealing tape Gel thermometer strips Micropipettes and elongated plastic tips Vaseline Syringes
1. Buffers and solutions
Acrylamide: Methylenebisacrylamide (29:1) (%, m/V)
Ammonium persulfate (10%, m/V)
Ethanol
6X gel loading buffer
KOH/Methanol
Silicification solution (e.g. Sigmacote or Acrylease) (optional or not)
5X TBE electrophoresis buffer
TEMED
2. Nucleic acids and oligonucleotides
DNA Sample
3. Specialized equipment
Binders or paper clips made of tin (6-8, 2 inch/5 cm wide)
Electrophoresis unit, glass plates, combs and spacers
Gel Sealing Tape
Gel temperature strips (optional or not)
Micropipettes and elongated plastic tips
Vaseline
Syringe (50 ml)
II. Methods
Setting up the electrophoresis apparatus and preparation of gel solution
1. Clean the glass plate and spacer with KOH/methanol if necessary.
2. Wash the glass plate and spacer with a warm detergent solution, then rinse well, first with tap water and then with deionized water. The edge portion of the glass plate should be held or handled with gloves so that grease from the hands is not left on the working surface of the glass plate. Rinse the glass plate with ethanol and set it aside to dry.
3. (Optional) One side of one of the panes should be treated with a siliconizing solution (e.g., Sigmacote or Acrylease): In a chemical fume hood, place the panes on a wad of paper towels, pour a small amount of siliconizing solution onto the surface, spread evenly with Kimwipes paper towels, rinse with deionized water, and dry with paper towels.
4. Install glass and spacer:
(1) Place the larger (non-notched) glass plate flat on the lab bench and place the spacers parallel to the edges of the glass plate on both sides.
(2) Apply a light coating of petroleum jelly to help keep the spacer in place during subsequent operations.
(3) Place the inner plate (notched glass plate) on top of the spacer.
(4) Clamp the glass panels together with clips or tin paper clips and seal the entire glass panel tightly on both sides and the bottom with gel sealing tape to form an impermeable seal.
5. Calculate the volume of gel required based on the size of the glass plate and the thickness of the spacer. Prepare a gel solution of suitable concentration.
6. (May or may not be done.) Place the desired amount of polyacrylamide: methylenebisacrylamide solution in a clean flask with side arms, add a magnetic stirrer, and evacuate for degassing. Degas gently at first and rotate the flask while degassing until no more bubbles escape.
Filling the gel
7. The following procedure should be performed with gloves on a tray to avoid spilling acrylamide:methylenebisacrylamide solution on the bench. The action should be rapid and completed before the polymerization of the acrylamide.
(1) Add 35 μl of TEMED per 100 ml of Acrylamide: Methylenebisacrylamide solution and mix the solution by gently swirling.
(2) Aspirate the gel solution with a 50 ml syringe, swivel the syringe and purge the air from the syringe. Insert the needle of the syringe into the space between the two glass plates and inject the acrylamide solution to almost fill the space.
(3) Place the glass plates diagonally against the test tube rack at an angle of about 10° to the laboratory bench.
8. Immediately insert a suitable comb into the gel, being careful not to leave air bubbles under the teeth of the comb. The tip of the comb should be slightly above the top edge of the glass plate. Clamp the comb in place with a tin paper clip. If necessary, completely fill the mold with the remaining acrylamide solution. Verify that no acrylamide solution leaks out of the mold.
9. Polymerize the acrylamide at room temperature for 30 to 60 min. If gel retraction is evident, add additional acrylamide: Methylenebisacrylamide solution.
10. When polymerization is complete, wrap the comb and the top of the gel in 1X TBE-impregnated paper towel. Then seal the whole gel with Saran Wrap film and store at 4°C until use.
11. When ready for electrophoresis, spray some 1X TBE buffer around the comb and on top of it and carefully remove the comb from the polymerized gel. Use a syringe to aspirate 1X TBE to flush the spiking wells. Remove the sealing tape from the bottom of the gel with a razor or scalpel.
Sampling and Electrophoresis
12. Place the gel in the electrophoresis tank and secure it with a large tin clamp around the inlet edge or with the clamp on the unit itself. The glass plate with the notch should be placed facing the buffer tank inward.
13. Fill the buffer tank with 5X TBE prepared electrophoresis buffer from the same batch used to prepare the gel solution. Remove any air bubbles hidden at the bottom of the gel with a bent Pasteur pipette or syringe needle.
14. Rinse the spiking wells with 1X TBE again using a Pasteur pipette or syringe. Mix the DNA sample with the appropriate amount of 6X Gel Loading Buffer and add to the spiking wells using a Hamilton syringe or micropipette with an elongated plastic tip.
15. Connect the electrodes to the power supply (positive to lower tank), turn on the power, and electrophoresis.
16. Electrophoresis until the standard reference dye has migrated to the desired location. Turn off the power, unplug and discard the electrophoresis buffer from the electrophoresis tank.
17. Remove the glass plate and use a scalpel or razor blade to remove the gel sealing tape. Place the glass plate on the lab bench (siliconized glass plate on top). Use a spacer or plastic wedge to pry up the corner of the top glass plate. Check that the gel is still attached to the bottom glass plate. Remove the top glass plate smoothly and remove the spacer.
18. Examine the position of the DNA bands in the polyacrylamide gel.