- 1). Obtain a detailed working protocol before initiating a gene analysis project, preferably from a scientist analyzing the same gene and DNA sample, using the same tools, reagents and machinery. Write out the protocol in technical, scientific terms. For example, write out the list of ingredients required for the reaction, as well as the exact concentrations, volumes, temperatures, incubation durations and termination parameters.
- 2). Gather all required reagents, such as enzyme mixes, buffers, cofactors (e.g., magnesium chloride) and DNA preparation chemicals. Also gather all tools and equipment, such as PCR pipettes and filter tips, as well as PCR strip-tubes and lids. Treat these with DNase-removal chemicals as necessary, or autoclave to inactivate these enzymes. Obtain training and usage licenses for operating PCR thermocyclers, and book a time slot for your experiment.
- 3). Purify, precipitate and quantify the DNA. PCR success relies heavily on the quality of DNA. Always maintain the highest DNA purity and integrity; otherwise, the experiment will fail regardless of how much magnesium chloride is used. Use DNase-removal chemicals to kill or inactivate DNase enzymes, which degrade and destroy DNA. Analyze the DNA by agarose gel electrophoresis if you are unsure of the quality.
- 4). Set up PCR reaction. Following the protocol, set up a master mix containing the PCR enzyme (polymerase), buffer, deoxyribonucleic triphosphates, primers, DNA and any other additives, such as inhibitors. Aliquot this into as many tubes as stated in the protocol.
- 5). Add magnesium chloride. Magnesium activates the polymerase and is added to reactions that consistently fail to produce any, or the desired level of, amplification. Since every DNA is different, there are no firm rules as to what concentration of magnesium should be added. This should be determined empirically by the experimenter.
- 6). Dilute a solution of magnesium chloride to 25 millimolar (mM) as a starting point. Label five tubes (containing the PCR reaction from Step 4) as follows: 2.5, 3.0, 3.5, 4.0 and 4.5 mM. To obtain 2.5 mM of magnesium chloride in a 20 microliter (uL) reaction, add 2 uL of 25 mM magnesium chloride. To obtain 4 mM of magnesium chloride in a 20 uL reaction, add 3.2 uL of 25 mM magnesium chloride, and so on. The reaction can now be run on a PCR thermocycler using cycling conditions as specified in the protocol.