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L A B O R A T O R Y
Part III: DNA Analysis by Gel Electrophoresis

Reagents Equipment & Supplies Shared Items
2% agarose gel
1X electrophoresis buffer
pBR322-BstNI markers
1 µg/ml ethidium bromide or 0.05% methylene blue staining solution
15 ml test tube, polypropylene
1-20 µl micropipet and tips
Staining tray or weigh boat 		Electrophoresis chamber
Electrophoresis power supply

Procedure

  1. Use a micropipet with a fresh tip to add 15µl PCR sample/loading dye mixture into your assigned well of a 2% agarose gel. (This will leave enough product if you intend to sequence the mt control region.) Expel any air from the tip before loading, and be careful not to push the tip of the pipet through the bottom of the sample well.
  2. Load 5 µl of the pBR322-BstNI size markers into one lane of gel.
  3. Electrophorese at 130 volts for 20-30 minutes. Adequate separation will have occurred when the cresol red dye front has moved at least 50 mm from the wells.
  4. Gel may be stained either with 1 µg/ml ethidium bromide for 10 minutes or 0.05% methylene blue (or proprietary) stain for 30 minutes, followed by 20-30 minutes destaining with water.
* If adding Bromophenol Blue loading dye to sample: do not add the loading dye to the entire sample, leave 10µl of sample without loading dye if you will be sending sample in for sequencing.
M I T O C H ON D R I A L  C O N T R O L  R E G I O N

2000, DNA Learning Center, Cold Spring Harbor Laboratory
Noncommercial, educational use only.
Kits available from Carolina Biological Supply Company.