The purpose of a PCR reaction is to amplify a targeted DNA region into millions of copies.  PCR helps to yield several copies of DNA in a few hours allowing analysis. Each PCR cycles approximately doubles the amount of DNA present in the reaction.

Real time PCR is a technique that measures the amount of fluorescence that occurs with each cycle of PCR. The amount of fluorescence is directly proportional to the DNA concentration.  The assay can also provide info such as the possible presence of inhibitors. 

It is important to know how much DNA is present in a sample to optimize downstream processing.

Extraction is a useful separation technique when there is a mixture of compounds in a solution that have similar polarities, but different solubilities. The three-step process of extraction can take advantage of different solubilities by introducing the mixture to different acidic and basic conditions.

Heating of the reaction to roughly 98°C is required to separate the template DNA strands from one another, thereby producing single strands that can pair to their complementary primer. At this temperature, the hydrogen bonds between base pairs break and the strands separate.

Taq polymerase doesn't require this temperature to be active, non-target sequences are not degraded during PCR, the dNTPs are already soluble, and the optimal annealing temperature for primers is actually much lower than 98°C. 

In an EMSA, a radiolabeled sequence of DNA that the protein of interest normally binds is incubated with the protein. This mixutre is then run on a non-denaturing gel. If the protein binds the radiolabeled DNA sequence, radioacitivity will be detected towards the top of the gel; however, if it does not bind, the radiolabeled DNA probe alone will run more quickly towards the bottom of the gel. 

The purpose of an electrophoretic mobility supershift assay (EMSA) is to determine if a certain protein binds a DNA sequence. EMSAs determine if a protein is "active", meaning that it is capable of binding its target DNA sequence. In an EMSA, a radiolabelled DNA fragment is incubated with cellular protein lysates, then run on a non-denaturing gel. Any protein-bound DNA fragments will migrate slower than unbound DNA fragments. When performing a supershift, one wants to determine if a specific protein binds a radiolabelled DNA probe by use of an antibody. If the antibody binds the protein-DNA complex, this will migrate even slower than the protein-DNA complex alone. 

A buffer system is required in a capillary electrophoresis system in order to supply the ions necessary to carry the electric current. Ions become depleted quickly and the buffer system will need to be replenished regularly.

The positive and negative electrodes, in combination with a high voltage power supply create the current. The source of the sample and destination of the sample will have opposite charged electrodes. Migration of the analytes through the polymer filled capillary will depend on the applied electric field and their size.

The detector of a capillary electrophoresis system will vary based on the instrumentation type. It is used to detect the size of the molecules and the speed at which they move through the matrix. Sizing methods and databases are then used for analysis.

Polymer serves as the separation matrix of the system. The viscosity of the polymer in combination with the electroosmotic flow of they system will separate molecules based on their size. 

Capillary electrophoresis allows for better reproducibility of liquid polymer throughout a capillary compared to slab gels. Much of the time gels are manually poured and uneven gel thickness can occur. 

Real time data viewing is possible on a capillary electrophoresis instrument's system computer. 

Higher sample consumption is common with slab gels. More sample is required to be loaded in each lane. If retesting is necessary, the sample must be prepared and loaded into a new gel. Very small quantities of sample are consumed in the injection step into the capillary. Samples can be easily retested through reinjection from the original sample vial.

Capillary electrophoresis has the possibility of being a completely automated process, including automated sample loading. This saves analyst time during sample prep, injection, and separation. Gels require manual sample loading and some instruments require gel handling for scanning or photography after the electrophoresis process. 

All of these methods require the immersion of the capillary end into the sample for introduction into the capillary electrophoresis system.

Gram staining is used to distinguish different types of bacteria in order to aid in their identification. Since bacteria are generally transparent, they must be stained before viewing under the microscope. Gram-positive microorganisms stain purple and Gram-negative microorganisms stain red or pink.

Acid fast stain is used to identify the causative agent of tuberculosis. White blood cells are differentiated after being stained with Wright's stain. Giemsa is used to stain blood cells and chromosomes. Tissues are stained with eosin.