Dye Lab – Write up in lab notebook

Purpose:

  1. To practice pouring gels, using micropipettes, loading dyes and running gel electrophoresis.
  2. To determine the movement of dyes in the gel matrix based on size and charge.

Materials:Make list of all materials and equipment used in the lab today.

Procedure: List steps as seen on board.

Observations:

Draw a picture of your gel. Draw the bands (in appropriate color) where they appear on gel. Set up as seen below:

+-

Data Chart: Create chart for 8 wells with dyes in proper order.

Well # / Dye Name / Dye Color / Distance traveled (mm) / Charge( + or -)

Conclusions:

  1. Name the positive dye with the largest molecules. How did you determine this?
  2. Which dyes were negative? How do you know?
  3. What dyes are present in the unknown? How did you determine this?
  4. Which dye traveled the fastest? How do you know?
  5. Where was the comb placed for dyes? Why is this?
  6. Where will the comb be placed for DNA? Why?
  7. What are the purposes of adding buffer to the gel rig?
  8. Complete the conversions below: 1,000 microliters (l) = 1 milliliter (ml)
  9. 10l = ______ml
  10. ______l = .015 ml
  11. 250 l = ______L
  12. Put the following volumes in order from largest to smallest:
  13. 2.5 ml, 250 l, .025 ml, 2.5 l
  14. 100l, .01 ml, 250 l, .015 ml
  15. You need to make 2% Cresol Red solution that is 10ml total in volume. Show work.
  16. How many microliters of dye would be needed to make this solution?
  17. How many milliliters of water would be needed to make this solution?

Dye Lab Procedure: Copy steps into lab notebook as you do lab today.

  1. Tape gel tray on both open sides 2 times.
  2. Place comb in the center of the tray.
  3. Pour agarose gel and let solidify.
  4. Pull comb straight up and out of agarose slowly.
  5. With black (negative) side up, load10 l of dyes 1to 8 in the wells from left to right.

1. Bromophenol blue

2. Janus green

3. Orange G.

4. Safranin O.

5. Xylene Cyanol

6. Cresol Red

7. Easy Green

8. Unknown

  1. Take tape off of tray.
  2. Place tray in gel rig. Be sure black line is on same side as black dot (neg)
  3. Pour buffer into the side of the rig until the gel is fully covered.
  4. Put lid on rig and plug electrodes into power supply.
  5. Plug in power supply and run rig at 150 Volts for 20 minutes
  6. Turn off power supply and unplug.
  7. Remove lid and pour gel carefully into plastic tray – making note of negative and positive ends.
  8. Measure distance dyes travelled, from the bottom of the well to the far end of the band.
  9. Record data in chart.
  10. Place gel in flask for recycling.