Methods: REP-PCR Running and Staining Box Fingerprint Gels (10-25-00)

Equipment

• Horizon 20x25 horizontal gel electrophoresis apparatus (Gibco-BRL)
• 30 tooth 1.0 mm thick comb (Gibco-BRL cat. # 11951-043)
• power supply
• pump (for buffer circulation)

Pre-made Solutions

• 1 kb ladder (0.5 µg/10 µl) (600 µl aliquots)
  • 30 µl Gibco/BRL 1 µg/µl 1 kb ladder stock
  • 100 µl 6X loading dye
  • 12 µl 1M NaCl
  • 458 µl ddH₂O
  • store frozen

• 6X Loading Dye
  • 25 mg bromphenol blue (0.25%)
  • 25 mg xylene cyanol (0.25%)
  • 1.5 g ficoll 400 (15%)
  • add ddH₂O to 10 ml, divide into 1 ml aliquots and freeze

• Ethidium Bromide Stain (0.5 µg/ml)
  • 1250 ml 0.5X TAE
  • 62.5 µl ethidium bromide (Sigma, 10 mg/ml)
  • store covered at room temp.

• Mix up 0.5X TAE
  • Add 20 ml 50X stock (Gibco-BRL) to 1980 ml diH₂O in a 2 l beaker, stir

Prepare Gel

• Place stirbar into a 500 ml orange cap bottle or flask
• Add 250 ml 0.5X TAE
• Add 3.75 g of SeaKem LE agarose to TAE, swirl to mix
• Microwave until agarose is fully dissolved
• Add thermometer and stir slowly until the agarose temperature is under 60°C if temperature is above 60°C gel tray will warp
• Pour gel into tray,
• After gel hardens, 10-15 minutes, move gel into cold room

• Connect pump tubing to gel box
• Tubing can easily be connected and disconnected by pressing in the metal tabs on the gel box
• Place remaining 0.5X TAE buffer in cold room (1750 ml) and allow buffer to cool with gel (can prepare the gels in the morning and run in the afternoon)

Prepare PCR samples for gel

• To each 25 µl PCR reaction add 5 µl 6X loading dye (27 PCR reactions X 5 µl dye/reaction = 135 µl dye)
• mix dye with the PCR reactions using a new yellow tip for each reaction, this eliminates having to mix dye and reactions in cold room

Load Gel

• Load 10 µl of 1 kb ladder (0.5 µg) into first lane on gel
• Load 10 µl of each 30 µl PCR reaction mix into next 13 lanes
• Load 10 µl of 1 kb ladder (0.5 µg) into lane 15
• Load 10 µl PCR reaction mixes into next 14 lanes
• Load 10 µl no template control into lane 29
• Load 10 µl of 1 kb ladder (0.5 µg) into last lane on gel, lane 30
• Run gel for 17.5 hours at 70 volts (start at 3 PM will be done at 8:30 AM)
• After gel has been running for 5 minutes, turn on the pump to recirculate the buffer

• Stain gel in 1.25 l of 0.5 µg/ml ethidium bromide in 0.5X TAE for 20 minutes
• No need to destain

Photograph gel and save digital image. We use a FOTO/Analyst Archiver (Fotodyne Inc., Hartland, WI) and integrate 150 images.

Ethidium bromide is mutagenic - wear gloves and labcoat when staining, rinse gel tray with diH₂O