Polyacrylamide gel electrophoresis

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! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)  
! Acrylamide Concentration(%) !! Optimal DNA Resolution (bp)  
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| 3.5 || 1000-2000
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| align="center" 3.5 || 1000-2000
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| 5.0 || 80-500
| 5.0 || 80-500

Revision as of 17:14, 17 June 2009

This procedure is useful for the separation of small pieces of DNA, small quantities of DNA, or applications where higher resolution than can be achieved with agarose gel electrophoresis is needed. Because it is very difficult to remove DNA from polyacrylamide gel, it is advised that this protocol should only be used for diagnostic purposes and not for size separation of useful fragments.

Contents

Materials

Reagents

  • 29:1 Acrylamide Solution
  • 10X TBE buffer
  • Ammonium Persulfate
  • TEMED
  • DNA solution
  • SYBR green
  • Bromophenol Blue

Equipment

  • Vertical electrophoresis chamber
  • Glass plates w/ spacers (which fit the chamber)
  • Casting holder
  • Pipettors

Procedure

1. Place Glass plates in casting apparatus
2. Add together the following to make 5ml of gel (0.75mm spacers)

  • 500µl 10X TBE solution
  • 35µl Ammonium Persulfate (10%w/v)
  • X mL 29:1 acrylamide solution (See the table under "notes" to determine the desired acrylamide concentration)
  • Y mL water (To make 5 mL)
  • 2µl TEMED

3. Pipet the acrylamide solution between the casting plates using a 5ml pipetor
4. Insert comb into the top of the gel and allow it to cure vertically for approximately 30 minutes
5. Combine the following for all DNA samples including the ladder

  • 10μL DNA solution
  • 1μL SYBR green (100X Dilution in DMSO)
  • 1μL Bromophenol Blue

6. Insert the gel into the electrophoresis chamber allong with the buffer dam

  • Make sure both the gel and the buffer dam seal
  • The wells on the gel should face the inside

7. Add 1X TBE to the space between the gel and tue buffer dam until the TBE fills the wells in the gel

  • No TBE should leak into the space outside of this chamber

8. Add 1X TBE to the outer chamber to the specified fill level. 8. Add DNA mix to wells.
9. Apply 80 volts and run for approximately 60 minutes


Notes

  • It is important to get the fill level right in the electrophoresis apparatus (please see the figure to the right).
  • For thicker gels (bigger wells) more gel will need to be made. The amount of gel necessary can be calculated volumetrically.
  • The voltage applied to the gel will vary according the the tube of gel you cast. The specified voltage is 1-8 volts/cm. Centimeters in this case specifies the length of the gel from top to bottom (i.e the direction the DNA will travel).
  • To determine the final acrylamide concentration for your application use the following table. For example: if I had a 40% acrylamide stock solution and I wanted 5mL of gel with a final concentration of 12% acrylamide, then I would add 5*(12/40)=1.5mL of acrylamide solution to make my gel.
Acrylamide Concentration(%) Optimal DNA Resolution (bp)
align="center" 3.5 1000-2000
5.0 80-500
8.0 60-400
12.0 40-200
15.0 25-150
20.0 6-100

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