How to Troubleshoot the Electrophoresis Process

Electrophoresis is a method of separating DNA or protein fragments by size. The gel is placed in an electrophoresis box and subjected to opposing charges. DNA carries a uniform negative charge, and so it naturally migrates towards positive charge and away from negative charges. Smaller fragments migrate further because they have an easier time maneuvering through the gel matrix. Proteins do not have uniform charges or shapes, and so they must first be subjected to a detergent, such as SDS, or Sodium Dodecyl Sulfate. The detergent uncoils the proteins and gives them a uniform negative charge.

Things You'll Need

  • Electrophoresis equipment and standard reagents
  • PCR polymerase chain reaction machine or microbial amplification vector
  • SDS-PAGE standard reagents
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Instructions

  1. Troubleshooting DNA or Protein Electrophoresis

    • 1

      Thoroughly wash the electrophoresis equipment. Rerun the experiment. If this fixes the problem, then the equipment or the technician's gloves may have been contaminated with an enzyme that degraded and the product, or another substance that inhibited the reactions.

    • 2

      If the experiment fails again, run another electrophoresis gel using different electrophoresis equipment. If this fixes the problem, then discard the faulty equipment.

    • 3

      If the experiment still does not work then rerun the experiment with different reagents such as buffer, polyacrylamide, dye and so forth. If this fixes the problem, one of the electrophoresis reagents may have been contaminated or otherwise faulty.

    Procedures Specific to DNA or Proteins

    • 4

      Repeat the procedures -- PCR, bacterial amplification, etc. -- that yielded the DNA sample in the first place, if you are getting a blank gel. If you are getting bands of the wrong size, then redo the digestion steps; for example, use restriction enzymes to isolate the desired DNA fragment; splice the receiving plasmid, if any and ligate the products. If this yields the correct results, then the problem was with the procedures that produced the DNA sample; not with the electrophoresis gel.

    • 5

      Repeat the experiment using uncut DNA. If this produces the predicted results, then the problem was with the digestion enzymes or procedures.

    • 6

      Repeat the procedures such as cell lysis, and so forth, that yielded the protein sample in the first place, if you are getting a blank gel.

    • 7

      If you are getting protein bands of the wrong size, then redo the SDS steps, being sure to heat the sample to allow the SDS to bind. If this yields the correct results, then the problem was with the procedures that produced the sample, not with the electrophoresis gel.

    • 8

      Repeat the experiment without the SDS. If this produces the predicted results, then the problem was with the SDS or associated procedures.

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