Restriction enzymes and electrophoresis

Protocol

Materials

  • Agarose (0.8%)
  • Distilled water
  • SYBER green DNA stain
  • λDNA (0.1 μg/μl)
  • Loading dye
  • 2xRestriction buffer
  • Restriction enzymes
  • EcoRI
  • BamHI
  • HindIII
  • 1xTris /Borate/EDTA (TBE) buffer
  • Micropipettes/tips
  • Centrifuge
  • Water bath (37oC)

Steps

I. Set Up Restriction Digest

  1. Use a permanent marker to label four 1.5-ml tubes, in which restriction reactions will be performed:

    • B = BamHI
    • E = EcoRI
    • H = HindIII
    • — = no enzyme

  2. Use the matrix below as a checklist while adding reagents to each reaction. Read down each column, adding the same reagent to all appropriate tubes. Use a fresh tip for each reagent. Refer to detailed directions that follow.

    Tube λDNA Buffer BamHI EcoRI HindIII H2O
    B 4μl 5μl 1μl - - -
    E 4μl 5μl - 1μl - -
    H 4μl 5μl - - 1μl -
    - 4μl 5μl - - - 1μl

  3. Set the micropipette to 4μl and add λDNA to each tube.

  4. Set the micropipette to 5μl and add buffer to each tube.

  5. Use fresh tips (change each time) to add 1μl of the appropriate enzyme
    (e.g. BamHI to the "B" tube).

  6. Use a fresh tip to add 1μl of the water to the "-" tube

  7. Close the tube tops and gently mix (tapping using your finger); then centrifuge briefly to collect the reagents at the bottom of the tube.

  8. Incubate the tubes on a 37oC water bath for at least 10 minutes.

II. Set Up Electrophoresis

  1. Seal the ends of the gel-casting tray, and insert well-forming comb. Place the gel-casting tray out of the way on the lab bench so that agarose poured in next step can set undisturbed.
  2. Carefully pour enough agarose solution into the casting tray to fill to a depth of about 5 mm. Gel should cover only about one-third the height of comb teeth. Use a pipette tip to move large bubbles or solid debris to the sides or end of tray while gel is still liquid.
  3. Gel will become cloudy as it solidifies (~10 minutes). Do not move or jar casting tray while agarose is solidifying. Touch corner of agarose away from comb to test whether gel has solidified.
  4. When agarose has set, unseal ends of casting tray. Place tray on the platform of the gel box so that comb is at negative black electrode (cathode).
  5. Fill box with TBE buffer, to a level that just covers entire surface of gel.
  6. Gently remove comb, taking care not to rip the wells.
  7. Make sure that the sample wells left by the comb are completely
    submerged.

III. Load and run gels

  1. Add 1μl of loading dye to each restriction digest tube use a fresh tip each time.

  2. Use a micropipette to add 10μl of the digest reaction + loading dye to the gel. Load in the order "B, E, H, -".

  3. Add DNA ladder to the gel.

  4. Ensure the gel is properly oriented in the gel tank (the wells should be closest to the cathode/black end).

  5. Run the gel for 30 minutes at 150V. About 10 minutes after starting, ensure the loading dye band is migrating towards the positive (red/anode) end of the tank.

  6. Once the gel run is complete, follow instructor instructions for visualizing the gel under ultraviolet light.