Joyce:DNA electrophoresis

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Contents

Procedure

To make a 1% gel:

  1. Mass out 0.5 g of agarose
  2. Place the agarose in a 250 ml erlenmeyer flask
  3. Add 50 ml of 1X TBE to the erlenmeyer flask
  4. Heat in the microwave for 1 minute 20 seconds
  5. Let the melted agarose cool off (about 5-10 minutes)
  6. While wearing gloves, add 4 ul of ethidium bromide and swirl. CAUTION: ETHIDIUM BROMIDE IS A CARCINOGEN. Always wear gloves when handling it. Always allow ample time for the agarose to cool before adding it as it will vaporize.
  7. Pour into a well-taped gel case containing the appropriate sized comb (3 mm x 2 mm for standard operations, 5 mm x 2 mm if you want to load it up as much as possible)
  8. Allow about 15-20 minutes to polymerize

To run the gel:

  1. While wearing gloves, remove the tape
  2. While still in the gel case, place the gel into the appropriate electrophoresis box
  3. Slowly and gently, remove the comb
  4. If necessary, add enough 1X TBE to completely cover the wells of the gel
  5. Load samples somewhere towards the bottom of the well (BE CAREFUL not to penetrate the bottom of the well, but make sure you get all of the sample in the well!)
  6. Place the lid on the electrophoresis box
  7. Connect the box to the power source
  8. Let run for about 30 minutes at 100 V, 20 minutes at 100V

To stop running the gel:

  1. Turn the voltage all the way down
  2. Turn off the power source
  3. While wearing gloves, remove the gel and place it into a container
  4. Place the cover back on the electrophoresis box

Notes

  • To better resolve bands less than 1000 bp, increase the concentration of agarose up to 2%. At this concentration, bubbles after pouring the gel are a pain in my rump, push them aside using a tip
  • After microwaving the agarose, it's important to let it cool sufficiently (you should be able to touch it to your wrist for 10 seconds without it hurting)

Calculations

0.50 g / 50 ml = 1%

Solution

TBE

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