E6: Gel Electrophoresis

What is gel electrophoresis?

GEL ELECTROPHORESIS is a method of separating DNA fragments by movement through a Jello-like substance called AGAROSE.

What is agarose?

1. AGAROSE is derived from a seaweed polysaccharide 2. Agarose gels form small pores that act as sieves to separate DNA based on size

How do smaller DNA molecules move through agarose gel?

Smaller DNA molecules move through the pores faster and easier than larger molecules.

What is the function of loading wells?

The loading wells allow for precise insertion of PCR PRODUCTS into the gel.

How does DNA move during gel electrophoresis?

Negatively charged DNA molecules move away from a negative electrode (-) and toward a positive electrode (+).

How does DNA migrate through the gel?

DNA migrates through the gel in a single vertical lane.

What factors influence the speed of DNA movement through the gel?

1. The voltage of the electrical field 2. The concentration of agarose 3. The size of the DNA molecule

Which factor most importantly influences DNA movement speed?

The size of the DNA molecule most importantly influences the speed of movement.

How is DNA visualized in an agarose gel?

1. A fluorescent stain is added to the gel 2. The fluorescent stain binds DNA 3. The fluorescent stain fluoresces under UV LIGHT or blue light 4. DNA appears as a horizontal line or band on the agarose gel

How do larger DNA fragments move compared to smaller fragments?

Larger DNA fragments do not move as fast as smaller fragments.

What is the relationship between DNA fragment size and distance migrated?

There is a nonlinear relationship between the size of the DNA fragments and the distance migrated.

What is a DNA ladder and why is it used?

1. A DNA LADDER is a sample with known fragment sizes 2. A DNA ladder should always be run to identify the size of experimental bands

What does gel electrophoresis allow?

Gel electrophoresis allows for differentiation of DNA by size.

What are the key elements for gel electrophoresis?

1. PCR PRODUCTS (DNA) 2. DNA LADDER 3. AGAROSE GEL 4. DNA STAIN 5. RUNNING BUFFER 6. LOADING DYE 7. ELECTROPHORESIS SYSTEM

What is the purpose of PCR PRODUCTS in this lab?

The purpose of this lab is to visualize the PCR PRODUCTS or amplified DNA from the bacterial samples.

What is a DNA ladder?

1. A DNA LADDER is a cocktail of DNA fragments with pre-determined sizes 2. The ladder is also called a DNA MARKER 3. The DNA ladder serves as a reference tool for estimating band size

How is a DNA ladder used?

The DNA ladder is loaded alongside experimental samples.

How is an agarose gel prepared?

1. Agarose powder is dissolved into running buffer 2. The agarose solution is boiled until the solution becomes clear 3. After slightly cooling, the solution is poured into a casting tray with combs 4. The solution solidifies and forms the AGAROSE GEL

What happens to DNA in an agarose gel?

1. DNA migrates through the gel 2. DNA forms separate bands based on size 3. The DNA band size correlates to length in BP

What is the function of DNA stain?

A DNA STAIN is added to the agarose gel to visualize DNA under UV LIGHT or blue light.

What are the two primary methods of DNA staining?

1. PRE-STAIN 2. POST-STAIN

When is DNA stain added in PRE-STAIN and POST-STAIN methods?

1. PRE-STAIN: DNA stain is added to the agarose gel mixture after melting 2. POST-STAIN: The gel is incubated in a stain solution after GEL ELECTROPHORESIS

What is the function of running buffer?

1. Running buffer is a conductive liquid 2. Running buffer allows the DNA to migrate through the agarose gel

What are the components and functions of loading dye?

1. A visible dye indicates how far the DNA has run on the gel 2. Glycerol is denser than the buffer 3. Glycerol ensures that samples fall into the loading wells rather than float back out

What happens in the electrophoresis system?

1. Running buffer and the agarose gel are placed into the chamber of an ELECTROPHORESIS SYSTEM 2. After loading the samples, an electric current is applied 3. The electric current moves the negatively charged DNA toward the positive end of the system

What happens without an electric field or if the field is reversed?

1. Without the electric field, the DNA will not migrate through the agarose gel 2. If the electric field is reversed, the DNA will run in the opposite direction 3. The DNA will move toward the top of the gel 4. The DNA will eventually exit the gel

How does DNA move in a gel?

DNA that was loaded into each well migrates in a single vertical lane toward the positive (+) charge.

How does DNA appear after gel electrophoresis?

1. DNA becomes separated by size due to GEL ELECTROPHORESIS 2. Separated DNA appears as bands in the gel 3. Bands are clearly defined bright lines in the gel

What does the DNA ladder contain?

1. The DNA LADDER contains multiple bands in one lane 2. Each band represents a pre-determined length of DNA

What is the function of the DNA ladder?

1. The DNA ladder can be used as a reference tool to estimate DNA size for each of the experimental samples 2. The product information should be referred to for specific band sizes

How are primer dimers formed?

1. PCR REACTIONS are set up with an excess of primers 2. Some primers bind to each other instead of binding to the DNA 3. Primer binding to each other creates PRIMER DIMERS

How do primer dimers appear on the gel?

1. Primers are approximately 25 BP long 2. Excess primers appear as fuzzy bands on the bottom of the gel at approximately 25–50 BP 3. The appearance of primer dimers is normal and expected

What is a single PCR gel?

A single PCR gel contains only one amplified PCR in each lane.

What is a duplex PCR?

Both the bacterial barcoding gene and the 16S rRNA FRAGMENT from other bacteria were amplified in the same PCR reaction.

How many gels are needed in a single PCR gel and duplex PCR gel?

1. A separate gel needs to be run for each DNA type 2. Both targets are visualized in the same PCR reaction

How do bands appear in a single PCR gel and duplex PCR gel?

1. One amplified PCR appears in each lane 2. Two bands should appear in the same lane if there is presence of other organisms in the solution

What does one band mean in a duplex PCR gel?

Only one band appears if the single bacterial species is uninfected.

What must be checked first when interpreting gel electrophoresis results?

1. The DNA LADDER, positive (+) bacterial control, negative (-) bacterial control, and positive (+) DNA control should produce bands of expected size 2. The water lane should be empty

How is the DNA ladder used to read the gel?

1. Experimental sample bands should be compared to reference bands in the DNA LADDER 2. The location of the bands in the gel is used to estimate sample size

What should be present in the positive controls?

1. The positive (+) bacterial control and positive (+) DNA control should both have the primer band and bacterial band present 2. In a DUPLEX PCR, the primer band and bacterial band appear in the same lane 3. In a standard single PCR, the samples are loaded into separate gels

How should DNA ladder bands appear?

The DNA LADDER BANDS should be clearly present and separated.

What should be present in the negative controls?

1. The negative (-) bacterial control should have a primer band 2. The negative (-) bacterial control should not have a bacterial band 3. The negative water control should not have any band or smudge

When are gel electrophoresis results considered valid?

1. If all controls worked, the results of the experiment are valid 2. If the results are valid, the experimental bands can be analyzed

What can affect interpretation of results?

1. Unexpected control results can affect interpretation 2. A band in the water lane indicates unexpected results

What are the main supplies and equipment used in gel electrophoresis?

1. Electrophoresis tank & power supply 2. Standard electrophoresis casting tray and comb 3. Microwave or hot water bath 4. Imager 5. Electronic balance 6. Squirt bottle or spray bottle with 70% ethanol 7. Gloves 8. Oven mitt 9. Eye protection 10. Weigh Dish 11. 1X Running Buffer (TAE or TBE) 12. 500mL flask 13. Graduated cylinder 14. Rack for 0.2mL PCR tubes 15. 20 μL pipette 16. 20 μL pipette tips 17. Waste cup for tips 18. Sharpie 19. Parafilm (optional) 20. Lab tape (optional)

What is included in a standard electrophoresis system?

A standard electrophoresis system includes a tank to hold buffer, and (-) and (+) electrodes that are plugged into a power supply.

What is the function of the casting tray and comb?

Used to cast gels. Before pouring the gel, the ends should be leak-proof, and a comb should be added.

What is the function of the microwave or hot water bath?

The microwave is used to melt the agar solution before casting into a gel.

What is the function of the imager?

A transilluminator, geldoc, or other imaging system to image and photograph the gel.

What is the function of the electronic balance?

A balance is needed to weigh the amount of agarose powder to make a gel.

Why is 70% ethanol used?

70% ethanol is used to clean the workspace before and after experiments.

Why are gloves used?

Gloves are used to protect both the scientist and sample from contamination.

What is the function of the oven mitt?

A waterproof, heat-proof oven mitt is used to protect the scientist from hot glass and steam after microwaving the agarose.

Why is eye protection required?

UV light can damage the scientist’s eyes.

What is the function of the weigh dish?

For weighing agarose powder on the electronic balance.

What is the function of 1X running buffer?

Running Buffer allows the gel fragments to migrate through the gel. It is a concentrated solution and needs to be diluted prior to use.

What is the function of the 500mL flask?

Used to boil the agarose powder and running buffer in the microwave to make molten agarose gel.

What is the function of the graduated cylinder?

A graduated cylinder is used to dilute the running buffer, and to transfer buffer to the electrophoresis tank.

Why is a rack for 0.2mL PCR tubes needed?

PCR tubes are small, it is necessary to have a tube rack so they are not lost.

What is the function of the 20 μL pipette?

Pipettes are used to move accurate and precise amounts of liquid from one place to another.

Why should each sample use separate pipette tips?

Tips should be changed between each sample to avoid cross contamination.

What is the function of the waste cup for tips?

Keeping all waste in one area until the end of the experiment increases efficiency.

Why is a Sharpie important?

It is extremely important to label all tubes and samples.

What is the function of parafilm?

1. Can be used to cover the agarose mixture in the flask while melting to prevent evaporation 2. It can also be used to mix PCR products with loading dye

What is the function of lab tape?

Can be used to seal off the ends of a casting tray before adding warm agarose.