DNA Electrophoresis (Lisa Hellinger, Shayleen Scantlin)

• • 5 - microtubes with blue, red, yellow, green, clear solution

  • 1 - .5 - 10 ul pipette

  • 1 - 5 - 50 ul pipette

  • 3 - empty microtubes for each student

  • 1 - tray of practice pipette tips

  • 1 - microtube of loading dye

  • 1 - practice gel submerged in tray of water

Pipetting Practice:

• 1. Send one team member to collect materials

  2. Using the small pipette, set it to the required setting and add the following: CAUTION! Change pipette tips between different solutions to avoid contamination. Place used tips, to be washed and saved, in the plastic cup. Do not place solutions on bottom of the microtube or to near the lid. “Hang” the droplets on the side of the wall without letting them touch.

     1. 4 ul of yellow solution to all microtubes. (This simulates the DNA)

     2. 5 ul of red solution to all microtubes. (This simulates the buffer)

     3. 1 ul of clear solution to only one microtube. (This simulates an enzyme)

• 1. Centrifuge your microtubes in order to mix the contents. CAUTION! Make sure the centrifuge is balanced or it will break! When you perform the lab the reaction between the DNA and the restriction enzymes will not begin until you centrifuge and place them in the waterbath.

  2. Add 2 ul of loading dye and centrifuge again to mix in the dye.

  3. How many total ul were added to your microtube? ________

  4. Set your large pipette to 10 ul.

  5. Withdraw 10 ul from your tube. Place the pipette tip at the bottom of the tube and very slowly withdraw the sample. The trick to this is to not let the tip of the pipette tip come off the bottom of the the tube even after you have released your thumb. The final drop will be pulled into the tip at the last second. If you pull the tip away too early you will get air. There should be no air bubble at the end of your tip.

Loading Practice:

• 1. When you have mastered step seven practice loading your sample into the gel.

NO NO YES!

• 1. Using two hands, steady the pipette over the well you are going to load. Expel any air in the end of the pipette before loading the DNA sample.

  2. Dip the pipette tip through the surface of the buffer, position it just inside the well, and slowly expel the mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the bottom of the well. Be careful not to puncture the bottom of the well with the pipette tip or re-aspirate your sample up into the pipette by letting your thumb release the plunger.

  3. When you are finished rinse out your microtubes. Leave the lids open, and place in the container designated for used microtubes and place practice gel on cart to dry.

Investigation Procedure:

Materials Needed:

• 250 mL of TBE Buffer

• 2.85 L of Distilled water

• 1% agarose solution

• Water bath or microwave

• Flask

• CarolinaBLU stain

• comb

• gel box

• DNA samples

• masking tape

• gel electrophoresis chamber

• power supply

• needle point pipets

• light box

Preparing the Agarose Gel

• Measure 1.25 g Agarose powder and add it to a 500 ml flask

• Add 125 ml TAE Buffer to the flask. (the total gel volume well vary depending on the size of the casting tray)

• Melt the agarose in a microwave or hot water bath until the solution becomes clear. (if using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask).

• Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly.

• Seal the ends of the casting tray with two layers of tape.

• Place the combs in the gel casting tray.

• Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white).

• Carefully pull out the combs and remove the tape.

• Place the gel in the electrophoresis chamber.

• Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.

Loading the Gel

• Add 6 ml of 6X Sample Loading Buffer to each 25 ml PCR reaction

• Record the order each sample will be loaded into the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder.

• Carefully pipette 20 ml of each DNA sample into separate wells in the gel.

• Pipette 10 ml of the DNA ladder standard into at least one well of each gel.

Running the Gel

• Place the lid on the gel box, connecting the electrodes.

• Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)

• Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes!

• Let the power run until the blue dye approaches the end of the gel.

• Turn off the power.

• Disconnect the wires from the power supply.

• Remove the lid of the electrophoresis chamber.

• Using gloves, carefully remove the tray and gel.

Movies:

References:

BioRad DNA Electrophoresis Video

Bozeman DNA Fingerprinting Video