I'm looking for some simple molecular biology lab experiments for under-graduates.
(All I have in the lab is light microscopes, micropipettes, aerobic incubator, spectrophotometer, and other tools...)
Any suggestions (of experiments and materials needed) to make my sessions more beneficial and interesting?
Hello Dalal Ghali
The standard molecular biology techniques include isolation and quantification of DNA, RNA and/or proteins, restriction digestion, sub-cloning, ligation, PCR, cDNA synthesis, separation by gel electrophoresis, Southern-blot, Western-blot, Northern-blot, and cloning.
For undergraduates, who may plan for simple experiments in molecular biology such as extraction of DNA from sources such as blood, body fluids, frozen tissue sections, mammalian cells, etc. Manual methods as well as commercially available kits may be used for DNA extraction. DNA extraction techniques include organic extraction (phenol–chloroform method), non-organic method (salting out and proteinase K treatment), and adsorption method (silica–gel membrane).
If your budget does not permit you to purchase DNA extraction kit, I would suggest you include the organic extraction (phenol–chloroform) method (protocol is easily available online) as one of your lab experiments.
You may assess the quality and yield of DNA by spectrophotometry or by gel electrophoresis. Spectrophotometry involves estimation of DNA concentration by measuring the amount of light absorbed by the sample at specific wavelengths. Absorption peak for nucleic acids is at ~260 nm. A 260/280 ratio of ~1.8 is generally accepted as pure for DNA.
Once you have DNA in hand, the next experiment you may plan is Polymerase Chain Reaction (PCR) which is a robust technique to selectively amplify a specific segment of DNA in vitro. For this purpose, you will require a thermal cycler which is not very expensive. PCR involves three main steps: (1) denaturation of dsDNA template at 92–95°C,
(2) annealing of primers at 50–70°C, and
(3) extension of dsDNA molecules at approx. 72°C.
These steps are repeated for 30–40 cycles.
Also, you will have to procure various chemical components required for PCR which includes MgCl2, buffer (pH: 8.3–8.8), dNTPs, PCR primers, target DNA, and thermostable DNA polymerase. You may select a target sequence which is the sequence within the DNA template, which will be amplified by PCR.
The PCR product after the completion of PCR can be visualized by running the sample on agarose gel. While preparing agarose gel, it is mixed with a fluorescent agent such as EtBr or SyBR green. Once the electrophoresis is completed (the dye front reaches the end of the gel) one can visualize the gel under UV using a gel doc system. DNA (PCR product) intercalates with the fluorescent dye and gives emission under UV light.
So, you may plan for three experiments namely,
DNA extraction,
Polymerase chain reaction that will selectively amplify a specific segment of DNA in vitro, and
Agarose gel electrophoresis.
These experiments will be interesting, and your students will gain considerable experience in basic molecular biology.
You will need to have the following equipment in the lab.
1. A refrigerator and at least one -20 degree C freezer to store the necessary chemicals, especially required for PCR.
2. Magnetic stirrer,
3. Vortex mixer,
4. Table-top centrifuge,
5. Micropipettes for PCR,
6. Thermal cycler for PCR,
7. Electrophoresis system,
8. Gel documentation system which is a device used to display DNA fragments after electrophoretic run (agarose gels typically stained with ethidium bromide or SYBR green).
9. PCR reagents including the primers.
Good Luck!
You could carry out mutagenic screens for drug resistance (number of colonies from parent population vs chemically mutagenized population). If you have access to an absorption spectrophotometer you could then do drug metabolism kinetics.
I really can't understand how you can deal with molecular biology with a light microscope !?
Cold Spring Harbor is a great source for information about a large number of molecular biology experiments.
If you have no autoclave, you can accomplish something similar by baking glassware in the oven with a foil cover and by boiling liquids 20 min in a pressure cooker. With sterile liquid medium, baked glassware, a shaking incubator and a spectrophotometer, you can experiment with growth conditions for bacteria or yeast, measuring absorbance over time. For example, you can vary pH, sugar, salt, temperature, etc. You could even add natural substances like the juice from crushed plants.
If you have or can purchase glass petri dishes, you can do mutagenic screens for nutritional deficiencies, which were very interesting to me when I was in undergrad. You can buy minimal agar and supplement with nutrients and find colonies that will grow with or without certain nutrients. Above they suggest a chemical mutagen, or you can mutate with UV light.
In undergrad, with a low budget, we experimented with growth conditions of plants with the roots in water supplemented with various minerals. We tested growth conditions of bacteria on agar plates. We did Drosophila genetics experiments. You have to buy a wild type fly culture, a mutant fly culture, medium for the test tubes and cotton or foam plugs for the test tubes. You have to have a refrigerator or ice or an inhaled drug to make them sleep so you can look at them under the microscope. My professor built his own gel electrophoresis apparatus for 10% of the cost of a new one. I'm not sure where he bought the materials.
I think that regardless of the type of experiment, they can learn the mindset of science; they can learn to perform an experiment carefully (with good sterile technique, for example) and perform data collection and analysis. Best wishes.
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