BCHE 341 Lab

Lab 11: DNA Agarose Gel Electrophoresis

The goal of this laboratory is to prepare an agarose gel and run your products from the previous laboratory through the gel. DNA is inherently negatively charged (why?) and will naturally be attracted to a positive electrode. Thus, an electrical attraction will be used to move the DNA through the gel. DNA travels through agarose by size. Small bands move more quickly than larger bands.

1. Prepare a chart in your lab report that looks like this:

lane	what? (Sample Name)	Each Sample Volume	loading buffer Volume (6X)	Total volume loaded
1.	Hind III λ DNA molecular weight marker	5 ul	2 ul	7 ul
2.	Uncut pICE plasmid	20 ul	5 ul	25 ul
3.	Hind III/BAMHI cleaved pICE plasmid	20 ul	5 ul	25 ul
	PCR product (DNA) (6 samples per group)	20 ul	5 ul	25 ul
	PCR DNA marker	5 ul	2 ul	7 ul

 

2. WEAR GLOVES. Label the required number of tubes, as recorded in your chart above. Make sure you mark your tubes accordingly so you know what is in each one.

 

3. Agarose Gel Preparation. The quantities of the solutions you will need are given below depending on your gel apparatus. “large” gel apparatus:

150 ml 1X TAE buffer

1.5 g agarose

Visualization dye (amount TBA) “small” gel apparatus:

50 ml 1X TAE buffer

0.5 g agarose

Visualization dye (amount TBA)

MAKE SURE YOU WEAR GLOVES FOR THIS PROCEDURE

1% Gel: Weigh out 1.5 gram of agarose and transfer it to an empty 250 ml Erlenmeyer flask.  Add 150 ml of 1X TAE buffer into the flask containing the agarose.  Give the flask a swirl. (Pay attention while heating to avoid boil-over) Heat the flask in the microwave, bringing the solution to a gentle boil.  During heating, you will need to stop the microwave from time to time and swirl the contents (frequently) during the heating to allow the agarose powder to go completely into solution.  This should take just less than one minute to one minute-and-a-half.  Once you no longer see particles of agarose, set your flask down on a stack of paper towels, or something not quite as cool as the table top.

 

4. If the Erlenmeyer flask is cool to the touch (like a babies bottle), you are ready to pour the solution from Erlenmeyer flask to the gel-casting tray.

 

5. Make sure that the gel-casting tray is positioned appropriately in the gel box, so that it forms a container to pour the gel into (the TAs will show you how to do this). Position the comb in the outermost notch and one in the middle.

 

ADD YOUR VISUALIZATION DYE. Swirl until mixed.

 

Slowly pour the warm gel solution into the casting tray by pouring it into one of the corners on side opposite from the comb.  Let the agarose gel solidify at room temperature; this takes 15-20 minutes.

 

6. Set the gel box on the bench with the negative (black) electrode to the left, electrode connectors pointing away from you. Carefully remove the gel tray from the gel box. Reposition the gel tray by turning it so that the wells of the gel are towards the left side (negative, black electrode) of the box.

 

7. Pour 1X TAE buffer into the gel box by pouring it gently directly over the gel. Pour enough of the buffer into the chamber to make approximately a 1 cm layer of buffer over the gel.

 

8. Gently remove the comb by pulling slowly directly upward on the comb.

 

9. Load you samples into the wells of the gel. You should be able to see the wells clearly, and loading the samples should be considerably easier than loading samples into a SDSPAGE gel. DO NOT push the pipette tip through the bottom of the well.

  

10. Your instructors will assist you in plugging the gel box into the power supply and turning it on. The voltage will be set at 100 V (or maybe somewhat higher). Let the gel ‘run’ until the blue dye (bromophenol blue) has moved at least half 2-3 inches across the gel (50-60 minutes).

 

11. Turn off the power supply, detach the power leads and gently remove the gel from the casting tray.
12. WEAR GLOVES and UV GOGGLES OR A UV MASK. Take the gel over to the light box and visualize the gel. Take a picture of the gel in your report.

 

13. The gel should be disposed of in the biohazard waste container provided.

 

14. Make sure you clean up before you leave.

 

DRAW and write down your conclusions regarding what you see on your gel. Be specific – what are the approximate molecular weights of the bands you see (in nucleotide base pairs)?

DNA Agarose Gel Electrophoresis Laboratory Report

Fill in this chart describing the samples you prepared for electrophoresis, include a description of the results:

 

lane	what?	amount	loading buffer	amount loaded
2	Uncut	6 ul	2 ul	8 ul
3	Cut	6 ul	2 ul	8 ul
2	1	20 ul	5 ul	25 ul
3	2	20 ul	5 ul	25 ul
4	3	20 ul	5 ul	25 ul
5	4	20 ul	5 ul	25 ul
6	5	20 ul	5 ul	25 ul
7	6	20 ul	5 ul	25 ul

 

Highlighted yellow are the plasmid samples Lanes 2 and 3

Highlighted Blue are the DNA PCR samples Lanes 2-7

1. One/some lane(s) should have multiple bands (e.g. molecular weight markers), and the restriction endonuclease lane(s) should have a single band with another smaller (generally harder to see) band of a much lower molecular weight than the other bands.

Also include the results for PCR product electrophoresis in the table (See hand out for Lab#9), and complete the Lab#9 report as well. Draw what you saw in your gel in the space provided below – label the lanes 1, 2, 3, etc., so that they match the above chart. Write in the molecular weights of each band in your molecular weights lane, and the approximate molecular weight of each band you see in each lane.

 

2. Look at the lane that contains your pICE plasmid, uncut. What do you see? If you see multiple bands, why? Explain what the multiple bands of the DNA plasmid tell you about the possible structure/shape of that DNA.

 

3. Examine the lanes that contain the same plasmid, cut with HindIII and BamHI. What do you see? Explain what a single band seen for the DNA plasmid would tell you about the possible structure/shape of that DNA.

 

4. The DNA was attracted to a positively charged electrode. Why? Draw a piece of DNA below (at least 2 nucleotides), including ALL the charges, and circle the regions that are responsible for DNA’s attraction to a positively charged electrode.

 

5. Determine the Relative mobility for the different bands in the gel and plot the graph using proper log-scale graph paper. Find out the molecular weights (in nucleotide base pairs) of each band from the graph, if possible.