What is Gel Electrophoresis?
- A common laboratory technique used to analyze and identify the size and types of DNA fragments.
- Important applications include fingerprinting in forensics, paternity tests, medicine, and research.
Mechanism/How it Works
- DNA fragments are separated according to their size.
- There is a positive and a negative electrode at either end of the gel box.
- DNA fragments are negatively charged so they always move to the positive end, thus this electric current pulls fragments through the gel.
GelDoc DNA gel electrophoresis photograph stained with ethidium bromide, Sgroey, CC BY-SA 4.0
- Although they are all mobile, smaller fragments move faster than large fragments.
- When stained, one can see the bands created by these moving fragments.
- Each band represents a group of fragments of the same size.
- Therefore, the bands furthest away from the loading wells (at the negative end) or closest to the positive end are the smallest, and those that did not move much are the biggest.
- There is typically a DNA ladder - an already prepared mixture of known lengths used as a reference for the unknown fragments being tested.
Materials
- Gel box containing electrodes (anode and cathode)
- Power supply
- Transilluminator (e.g. UV or blue light)
- DNA samples
- DNA ladder (fragments with pre-determined lengths)
- Agarose Gel
- DNA Stain (fluorescent molecule binding directly to nucleic acids, must work with transilluminator)
- Running Buffer (conducts current, maintains reasonable pH, helps separate fragments)
- Loading Dye
- Electrophoresis System
Gel Electrophoresis, Mckenzielower, CC BY-SA 4.0
Sample Problem
A student investigates whether Sample C contains DNA from certain bacterial strains that infect crop plants. The student uses a PCR (polymerase chain reaction) test to amplify the expression of the sample’s genes.
After running the sample through agarose gel electrophoresis, the following bands from sample C are observed, in relation to a predetermined DNA ladder:
Adapted from Gel Electrophoresis, Mckenzielower, CC BY-SA 4.0
Based on the following information chart about what each length means, determine whether or not this sample contains bacterial strains. Specify which ones.
| Base Pairs/Band Size | Meaning If Band Is Present |
|---|---|
| 1,000 bp | Confirms that PCR worked and thus bacterial DNA is present |
| 900 bp | Plant Tissue DNA is present |
| 800 bp | Indicates fungal contamination |
| 700 bp | Bacterial Strain C is present |
| 600 bp | Bacterial Strain A is present |
| 500 bp | Bacterial Strain B is present |
| 400 bp | Environmental contamination |
| 300 bp | Harmless bacterium |
| 250 bp | Vector DNA |
| 200 bp | Disease-causing factor |
| 100 bp | No meaning |
Step-By-Step Solution:
- Compare sample C's bands to that of the DNA ladder: there is a lot of aggregation at 1,000 base pairs, some at 600 base pairs, 500 base pairs, and 250 base pairs.
- Compare the fragments present to their meaning: 1,000 base pairs confirms that bacterial DNA is present and the PCR worked. It is important to get that confirmation, but it does not contribute to our final answer. 600 base pairs indicates Strain A is present, 500 base pairs indicates Strain B is present, and 250 base pairs indicates vector DNA is present.
Answer: Yes, this sample contains bacterial strains, specifically Strains A and B.