DNA Sequencer

DNA Sequencer

SOP for Owl Nugeneration DNA Sequencing Unit

  1. With the thumbscrews of the Lower Buffer Chamber facing forward stand the Upper Buffer Chamber (UBC) in Lower Buffer Chamber (LBC) with red gasket of UBC facing forward.
  2. Stand gel cassette in the LBC and lean it against the gasket in the UBC. Put safety bar in place without tightening the thumbscrews.
  3. Place side clamps over the sandwich (UBC and gel cassette) as close to the top as possible with thumbscrews facing away from you. Tighten thumbscrews evenly - DO NOT OVERTIGHTEN.
  4. Close buffer drain ports by pressing in the steel tabs; spigot will pop out. Remove safety bar and pour buffer into the LBC. Replace safety bar; buffer level should be just below the safety bar. Tighten thumbscrews; this will push the safety bar up against the UBC.
  5. Open UBC safety lid by sliding to the left and lifting up. Pour buffer into the UBC until buffer is above notched gel plate. Close lid.
  6. Plug sequencer into power supply and prerun gel 30-60 minutes. This will warm the buffer and align the molecules in the gel.
  7. Turn power supply off and unplug sequencer from power supply.
  8. Open UBC safety lid and load samples into the spaces between the teeth. Close lid and reconnect to the power supply. Turn power supply on and run gel.
  9. After gel has finished running, turn power supply off and drain LBC and UBC. To open drain ports, push the white spigot in. Remove side clamps and the safety bar.

Otter Gel Caster:

  1. Clean glass thoroughly. Wash surface with ethanol and wipe with a dust free cloth (Kimwipe).
  2. Lay unnotched plate on the Otter gel caster.
  3. Lay spacer strips on both sides of plate, leaving the bottom open. Put a drop of water on unnotched plate to hold spacers in place.
  4. Place notched plate on top of unnotched plate, overlapping 1 inch.
  5. Warm gel burst pak to room temperature - make sure there aren't any crystals floating around in the large compartment of the burst pak.
  6. Roll gel pack on to itself, bursting the compartments, and mix by tipping back and forth.
  7. Cut a corner of the gel pack and slowly pour onto unnotched plate as the notched plate is being slid over the unnotched plate. If a bubble forms, slide plate back and slowly slide forward again.
  8. Once gel is poured, place binder clamps at cutouts over the spacers. Insert the comb so that the blunt end of comb is in the gel. Push comb in so that the oval cutouts are even with the top of the notched plate and the teeth are pointing away from the gel.
  9. Once the gel is polymerized, flood the comb with 1X TBE. Remove the comb and clean it, reinsert the comb so that the teeth are penetrating the gel. Push the comb in, leaving a place to insert a pipette for loading.

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