Question

Lab 9 – Molecular Biology

In this lab, you will prepare an agarose gel and use gel electrophoresis to compare the size of 2 dye molecules Methyl orange and Ponceau G. You will also analyze an “unknown” sample that contains a mixture of two dyes. Dye molecules with lower molecular weight or greater electrical charge will migrate faster through the gel, than dye molecules with greater molecular weights or lesser electrical charge.

Materials:

Mini gel electrophoresis chamber

6 Tooth comb

Aligator clips

9V Battery

(1) 100ml Beaker

Carbon fabric

3 Micro?pipettes

10X TBE Buffer

*Boiling water bath or microwave oven

Methyl orange

Ponceau G

Unknown #1

0.8% Agarose

*Water

Ruler

*You must provide

1. To prepare a 1X solution of TBE (Tris?borate EDTA) buffer, mix 10ml of the 10X TBE with 90 ml water in a 100ml beaker. Note: If the 10X solution has precipitated, place the bottle in a hot water bath and swirl until precipitant dissolves.

2. To prepare the agarose, loosen the cap on the bottle of 0.8% agarose and heat in the microwave or boiling water bath. Monitor the bottle closely and remove the bottle from the heat every 15 seconds and swirl the contents to mix. The agarose will become clear when melted, after about 40 – 45 seconds in a microwave

Note: Agrose gets very hot, very quickly. It is important to watch the container closely to prevent boiling over. Do not touch the container immediately after heating and always use a hot pad or towel when removing the bottle from the heat source.

3. Remove the bottle from heat and let cool to a safe handling temperature (it should still feel warm ~60°C).

4. Insert the comb into the slots in the gel compartment to mold the wells when the gel is formed.

5. Carefully pour the viscous agarose into the gel compartment of the mini?gel chamber. The container holds more agarose than what is needed to fill the chamber, so do not pour all the agarose in, only fill in between the two raised grooves on the bottom of the chamber.

Hint: The grooves are noticeable from above and from the side, about 1/4 inch from the top and bottom of the chamber. If you prefer, it may be helpful to be eye level with the chamber when pouring the agarose. When looking straight on, from a lateral view, you can see the height of the grooves and where to stop when pouring the agarose.

6. While the gel is still liquid, it is important to remove any air bubbles that may have been introduced while pouring.

Hint: Use a pipette tip to “poke” the bubbles or move them to the side.

7. Once the gel has solidified (10 –15 minutes) carefully remove the comb.

Hint: You may have to softly jiggle the comb out of the gel.

8. Once the comb is removed you will see six troughs, or wells, in the gel. This is where you will pipette the samples.

Note: The dyes have been mixed with a sucrose solution to increase their density and insure your sample will fall gently into the well. However, it is still important to run the gel relatively quickly so the dyes do not diffuse out of the wells.

                                                                                           

9. Cut 2 - 2” x 2” thick pieces out of the carbon fabric, one for the red clip and one for the black clip. Fold the carbon fabric so that you can drape it over the top and bottom edges of the gel chamber. Clamp the black alligator clip to the top (closest to where you loaded samples), and the red clip to the bottom .

10. Shake down the dye from the top of each tube so it collects at the bottom of the tube.

11. Load the dye samples from left to right following the order listed below in Table 1.

12. With a steady hand, pick up 20?l with the needle?point pipette (You should fill the narrow portion of the pipette to the bottom of the squeeze bubble.)

13. Expel any air that is at the tip of the pipette so that it is not introduced into the well. Remove excess dye off the outside of the pipette

14. Submerge the tip into the well and slowly dispense the dye sample into the well.

15. Repeat this process for each dye sample, including the unknown. Remember to use a clean pipette for each sample!

16. Using a clean needle-point pipette, carefully seal the dye into the wells by adding 1 small drop of agarose on top of the surface,

17. Carefully add pour enough 1X TBE buffer to completely submerge the gel, and have enough to run over and cover the bottom of the carbon paper.

18. Attach the other ends of the alligator clips to the 9V battery. The black clip is attached to the negative terminal. The Red clip is attached to the positive electrode.

19. Watch carefully for five hours. Note: If you let the experiment run too long, the dyes will move totally off the gel!

20. Once separation of the dyes has been achieved, disconnect the alligator clips from the battery.

21. Measure the distance from the wells to the center of the bands in millimeters and record this measurement in Table1.

22. Also in Table 1, record the number of bands visible for each dye sample.

23. Take a photograph of your well, and submit it, along with this worksheet (once you’ve completed it), as an attachment to the assignment dropbox for this lab.

Table 1. Electrophoresis of Dye Samples
Well	Sample	# bands	Migration (mm)
1	Ponceau G
2	Methyl Orange
3	Unknown 1

Questions

1. What is the basis for the electrophoresis of different dye molecules?
    (select all that apply)

Molecular weight

Length

Difference in charge

2. Which of the 2 dye samples (Ponceau G or methyl orange) has the greatest charge?

3. How did your results demonstrate this?

4. How many dyes are in the unknown sample? How can you tell?

5. Why is electrical current necessary to separate molecules using electrophoresis?

6. What is the charge(s) of the samples, including the unknown?

7. What do you think would happen if the molecules were the opposite charge?

8. What would happen if you attached to red and black electrode on the wrong sides?

Answer 1

1. Electrophoresis is the movement of molecules under the electric field influence. In electrophoresis bands are formed accirding to electric charge on the molecules and molecular size.

2. Ponceau G and methyl orange both have negetive charge but Ponceau G has a more negetive charge then methyl orange.

3. In experiment net more charge on the Ponceau G can be determined by the movement of dye in electric field and more negetive will move with greater velocity.

4. Types of dyes can be known by running gel. Different dyes move with different velocity. Types of band formed can be detected.