Basic Methods in Cellular and Molecular Biology

Accurate cell counting is an important step in many biomedical experiments. Scientists often need a known number of cells to get reliable, reproducible, and statistically relevant data. A hemacytometer is a common tool for this task because it uses two laser-etched grids to help count a small sample of cells under a simple light microscope.

The counting process begins by adjusting the sample concentration. This helps avoid trying to count too many cells or too few cells. A small aliquot, or...

Cell line health and re-seeding are key parts of keeping cells ready for biomedical experiments. Cell lines grow quickly, so they can be expanded for experimental analysis more easily than freshly isolated, or primary, cells. They are often cultured under similar conditions to primary cells, but they still need their own specific growth factor cocktails.

Because cell lines can overgrow fast, their culture must be monitored closely. Mutations that allow indefinite growth can also lead to rapid...

PCR uses thermocycling to make many copies of DNA. Thermocycling means repeating cycles of temperature changes at fixed time intervals. With a thermostable DNA polymerase and DNA building blocks called dNTPs, the reaction can produce numerous copies from a small DNA sample.

The PCR cycle has three main steps: denaturation, annealing, and elongation. During denaturation, heat disrupts the hydrogen bonds between DNA bases and separates the strands into single-stranded DNA. During annealing, the...

DNA gel electrophoresis separates DNA molecules using an electric field and an agarose gel matrix. The gel is made of agarose, which forms a porous network that helps DNA fragments move and sort by size. The negatively charged phosphates in the DNA backbone make the fragments migrate toward the anode, the positively charged electrode.

As the DNA moves through the gel, smaller fragments travel more easily than larger ones. This lets the fragments resolve into separate bands on the agarose gel.

SDS-PAGE is a common lab method for analyzing proteins by size. It uses sodium dodecyl sulfate, or SDS, an anionic detergent that gives proteins a more even charge after they are linearized. The proteins are then loaded into a polyacrylamide gel and separated with an electric field.

During the run, the electric field pulls the SDS-coated proteins toward the anode. Larger proteins move more slowly through the gel than smaller proteins, so the mixture separates by size. Protein standards with...

Bacterial transformation lets cells take up foreign DNA from their surroundings. Some bacteria can do this naturally. In molecular biology, researchers recreate the process in the lab by making pores in the bacterial cell membrane.

Cells that can take up DNA from the environment are called competent cells. In the lab, bacteria can be made competent before DNA is added. The heat shock method is one way to do this and then introduce plasmid DNA into the cells.

This method uses a calcium-rich...

Bacterial transformation can be done by making electrocompetent cells. In this process, foreign DNA enters bacterial cells after an electrical field creates small pores in the cell wall. The cells that can take up DNA from the environment are called competent cells. In the laboratory, scientists can produce electrocompetent cells for this purpose.

The procedure uses an electroporator and an electroporation cuvette. The video follows the steps for preparing electrocompetent cells and then...

ELISA is a protein detection test that uses antibodies and a color change to identify a target in a sample. ELISA stands for enzyme-linked immunosorbent assay. It is called an immunoassay because antibodies do the detecting.

The test is carried out in a microtiter plate with small wells. In many cases, the target protein coats the bottom of a well. Antibodies then bind to that protein through a series of incubation and washing steps. Many of these antibodies are conjugated, or linked, to an...

Plasmid DNA cleanup from bacterial cells is a lab method for separating plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA molecule that carries a specific DNA fragment. After transformation into a host organism, the plasmid is replicated and makes many copies of the DNA fragment under study.

The process follows three main stages. First, the bacterial culture is grown. Next, the bacteria are harvested and lysed,...

Purifying DNA from agarose gels lets students recover a separated DNA fragment after electrophoresis. The DNA is first isolated from the gel, then moved through a silica column for cleanup.

The process has three main steps: binding, washing, and eluting. DNA is believed to bind to silica when high salt creates a salt bridge. After that, the DNA is washed to remove impurities.

The final step is elution, which happens under low-salt conditions. These conditions disrupt the DNA-silica...

Western blotting is a lab method used to detect specific proteins in samples that have already been separated by electrophoresis. It helps scientists find a single protein within a complex mixture by using antibodies that recognize it.

First, the proteins are separated by SDS-PAGE, which stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then western transfer moves the proteins from the polyacrylamide gel onto a membrane. The membrane holds the proteins in their original...

Transfection is the process of putting genetic material into mammalian cells. The material can be DNA or double stranded RNA. DNA lets the cell make proteins using its own machinery, while RNA is used to reduce the production of a specific protein by blocking translation.

DNA and RNA act in different parts of the cell. Transfected RNA works in the cytoplasm. DNA must first reach the nucleus to work well. Once there, DNA may be expressed for a short time, or it may become part of the cell’s...

DNA ligase joins DNA fragments by forming a phosphodiester bond. The ligase enzyme carries out this reaction in cells and in the lab. It helps repair single-strand and double-strand breaks that can happen during DNA replication.

In molecular cloning, DNA ligase connects an insert with a vector, which is a carrier DNA molecule that can replicate target DNA in a host organism. A generalized ligation reaction follows a basic setup. The video also explains how sticky end overhang length can affect...

Restriction enzymes are bacterial proteins that cut DNA at specific sequences. They are also called endonucleases. In bacteria, these enzymes help defend against bacteriophages, which are viruses that infect bacteria. The bacterial DNA is protected because methyl groups are added, so the restriction enzymes cut the invading DNA instead.

The video also explains how restriction enzymes are named and how their recognition sites work. It describes the kinds of DNA ends that can be produced after...

Molecular cloning in bacteria uses a vector to carry recombinant DNA so it can replicate inside a host organism. The DNA fragment of interest, also called the insert, may come from a prokaryotic or eukaryotic specimen. A clear cloning strategy helps guide the work from the start.

The insert and the vector are cut with restriction enzymes, which are proteins that cut DNA at specific sites. After cutting, the DNA pieces are purified. The purified insert and vector are then joined by ligation, a...