Toronto/Lab Protocols/Digestion


Plasmid Digestion

2 – 3 hours

  • You may want to quantitate your miniprep or purified DNA to find the concentration of DNA. If it turns out that any of your samples are particularly concentrated or dilute, you can adjust the volumes in the digest accordingly.
Buffer for given enzymes ³
Enzyme EcoRI XbaI SpeI PstI
EcoRI EcoRI 2 EcoRI EcoRI
XbaI 2 2 2 3
SpeI EcoRI 2 2 2
PstI EcoRI 3 2 3
  • For a total volume of 10 μL ¹:
    • 7.0 μL Plasmid ²
    • 1.0 μL BSA
    • 1.0 μL Buffer
    • 0.5 μL Enzyme1
    • 0.5 μL Enzyme2
  • Gently vortex to mix the solutions and spin it down for 10 seconds to make sure there is nothing left on the side of the tube. Incubate for 1 hour.
  1. When doing a digestion in order to perform a subsequent gel extraction, increase the total volume, usually to 30 μL. That way you can maximize the amount of DNA you have.
  2. When doing a digestion in order to do a length check, conserve plasmid and use only 1-2 μL. Top off the volume with 6-5 μL ddH2O.

Agarose Gel Electrophoresis

Prepare Agarose Gel

  1. Add 80 mL 1X TAE and 0.64 g Agarose in a large beaker. Heat for 1.5 minutes in microwave until the Agarose is completely dissolved.
  2. Once solution is cool enough to hold on the palm of your gloved hand, add 3.25 μL of Ethidium Bromide (EtBr) in the fume hood.
  3. Swirl beaker to diffuse the EtBr, and then pour into the gel holder with the desired well markers.
  4. Wait ~30 minutes for gel to set and rinse out the beaker with hot water.

Loading Samples

  1. Have at least 1 well with 3 μL 1 kb Ladder, 2 wells for more accuracy
  2. Mix samples with 2 μL Blue Loading Dye before loading the whole volume into the gel. If possible, leave blank wells between samples and ladder.

Running the Gel

  1. Set the voltage to a constant 100 V and the time to 30 minutes. Press “Run”. Always turn off power immediately after the gel is finished.

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