Molecular Techniques and Methods

Polyacrylamide Gel Electrophoresis of Nucleic Acids
(Non-denaturing, Native PAGE)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent, N,N'-methylenebisacrylamide (Bis). The reaction is a free radical polymerization, carried out with ammonium persulfate as the initiator and N,N,N',N'-tetramethylenediamine (TEMED) as the catalyst.




MATERIALS AND SOLUTIONS

30% Acrylamide:Bis Solution (29:1)
Acrylamide ------------------------------------------ 29 g
Bis-acrylamide --------------------------------------- 1 g
Add distilled H2O to make a final volume of -------- 100 ml
  • Dissolve the acrylamide and Bis-acrylamide in distilled water for 1 hour.
  • Filter the solution through a 0.4 um filter to remove undissolved particles.
  • Store in a brown bottle at 4oC for 1 month.
  • Caution: Acrylamide is a neurotoxin!


    10% Ammonium persulfate (10 ml)
    Ammonium persulfate -------------------------------- 1 g
    Add distilled H2O to make a final volume of -------- 10 ml


    5 x TBE Buffer (1 liter)
    0.32 M Boric acid ----------------------------------- 10 g
    0.5 M Trizma base ---------------------------------- 30.25 g
    10 mM EDTA-Na2 --------------------------------- 1.86 g
    Add distilled H2O to make a final volume of ------- 1 liter
  • Adjust pH to 8.6.


    50 X TAE Buffer (100 ml)
    0.4 M Trizma base ------------------------------- 24.22 g
    10 mM EDTA-Na2 ------------------------------ 1.86 g
    Glacial acetic acid -------------------------------- 13 ml
    Add distilled H2O to make a final volume of ----- 100 ml
  • Adjust pH to 8.0.


    Ethidium Bromide Solution (10 mg/ml)
    Ethidium bromide --------------------------------- 100 mg
    Add distilled H2O to make a final volume of ----- 10 ml
  • Store in a brown bottle at 4oC.


    10 X Glycerol Dye Mix (10 ml)
    Glycerol ------------------------------------------ 6 ml
    0.2% Bromophenol blue -------------------------- 0.4 ml of 5% Bromophenol blue
    0.2% Xylene cyanol FF --------------------------- 0.4 ml of 5% Xylene cyanol FF
    5 X TBE (or 5 X TAE) ------------------------ 2 ml
    Distilled H2O ------------------------------------- 1.2 ml


    10 X Ficoll Dye Mix (5 ml)
    50% Ficoll type 400L (MW 400,000) ----------- 2.5 g
    0.14% Bromophenol blue ------------------------ 0.14 ml of 5% Bromophenol blue
    0.14% Xylene cyanol FF ------------------------- 0.14 ml of 5% Xylene cyanol FF
    5 X TBE (or 5 X TAE) ------------------------ 2 ml
    Add distilled H2O to make a final volume of ----- 5 ml




    PROCEDURES

    1. Prepare the gel apparatus.

    2. Mix the polyacrylamide gel solution in a beaker.

    4%
    5%
    6%
    7%
    30% Acrylamide:Bis (29:1)
    13.4 ml
    16.7 ml
    20 ml
    24 ml
    5 X TBE (or, 5 X TAE)
    20 ml
    20 ml
    20 ml
    20 ml
    Distilled H2O
    56 ml
    52.3 ml
    49 ml
    45 ml
    10% Ammonium persulfate
    750 ul
    750 ul
    750 ul
    750 ul



    3. Add 85 ul of TEMED (N,N,N',N'-tetramethylenediamine).

    4. Immediately, pour the gel mixture slowly into the previously prepared gel form, until the liquid level reaches the top of the upper glass plate.

    5. Insert the comb between the two glass plates and clamp in place with a small spring clip.

    6. Dribble a little extra gel mixture over the junction between the comb and the glass plates, using a pipette, to compensate for volume shrinking upon gelation and small leaks. Repeat as necessary.

    7. Let the gel stand 1-2 hours or overnight to solidify.

    8. Remove the comb by gently wriggling it and lifting out.
  • Wash unpolymerized acrylamide out of the wells by squirting with distilled water.
  • Remove the water from the wells.
  • Remove the spring clips and bottom space from the gel form.

    9. Attach the ears to the gel form.
  • Place narrow lines of high vacuum grease from each edge of the glass plate.

    10. Clamp the gel form to the gel cabinet with two large spring clamps.

    11. Fill the lower and upper tank with 1 X TBE or 1 X TAE.
  • The running buffer must be the same as the buffer in which the gel was cast.

    12. Use a bent syringe needle to perforate and remove air bubbles trapped in the space where the bottom spacer was originally located.
  • Remove any air bubbles from the wells of the gel by squirting with running buffer.

    13. Allow the gel to equilibrate with the buffer in the gel cabinet for 1-24 hours.

    14. Pre-electrophorese polyacrylamide gels for 2 hours, at a voltage of 3 volts/cm.
  • During pre-electrophoresis, the current will decrease and then become essentially constant.

    15. After the pre-electrophoresis is finished, turn off the high voltage.
  • Load the DNA samples (mixed with Glycerol Dye Mix or Ficoll Dye Mix) in the wells.
  • To get sharp bands, the DNA sample should be 1 mm or less in height after loading.

    16. Set the high voltage to the desired value and start the electrophoresis run.

    17. After the electrophoresis is finished, turn off the power supply.
  • Pour off the buffer in the buffer tanks.

    18. Remove the side binder clamps and pry the gel from the gel cabinet by placing a metal spatula between the back glass plate and the gel cabinet and twisting gently.
  • Separate the top glass plate from the gel.

    19. Place the glass plate holding the polyacrylamide gel in 100 ml of a 1 X TBE (or, 1 X TAE) solution and 15 ul of Ethidium Bromide Solution (10 mg/ml).

    20. Stain the gel for 15-30 minutes.

    21. Pour off the ethidium bromide-staining solution.

    22. Destain the gel 15-30 minutes in distilled water.

    23. Illuminate analytical gels with short wavelength UV light (254 nm) generated by Mineral-light model R-52.

    24. Illuminate preparative gels with long wavelength UV light (365 nm) generated by Mineral-light model R-56, to prevent nicking of the DNA/dye complexes.

    25. Photograph the gel




    NOTES

  • Polyacrylamide gels must be pre-electrophoresed before use, in order to eliminate charged impurities (assumed to be ammonium persulfate derivatives) from the gel matrix. If this is not done, the charged impurities will form complexes with the DNA fragments, changing their electrophoretic mobilities in a non-reproducible manner.

  • The polymerization reaction requires 24 hours to reach completion. For accurate, quantitative determination of DNA electrophoretic mobilities, polyacrylamide gel should be aged 24 hours.

  • When Joule heating becomes important, the temperature near the center of the gel is higher than the temperature near the edges. The resulting difference in the viscosity of the solvent causes the DNA bands to migrate faster near the center of the gel, giving a smiling effect when the same sample is loaded in all the wells.




    KIT INFORMATION




    REFERENCES


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