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Once you have your genetic samples, one of the first steps of PCR is to design the primers required to perform the PCR reaction.
The first step to proceed with a standard PCR is the primer design. You will need to determine which fragment of the DNA template you are looking to amplify, which will require you to know the DNA sequence of the template. The NCBI database, a web server with all DNA sequences known, is a good resource you can use to look for your sequence. Afterwards, you should design two primers, the forward and the reverse primer. Primer design is a critical step in a PCR protocol. The set of primers should flank the fragment you intend to amplify from the DNA template. The forward primer will anneal with 3’-5’ DNA strand and the reverse primer will anneal with the 5´-3’ DNA strand. Please refer to the diagram below.
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Many of the problems with PCR protocol are associated with wrong primer design. The most common mistakes in design include:
The primer length should be around 18-30 bp, the GC content close to 50%, and the melting temperature (TM) between 55ºC and 65ºC. In order to calculate melting temperature, you can use the following equation Tm = 2°C(A+T) + 4°C(G+C). Although most commercial DNA polymerases provide their own calculator and specific instructions for different conditions, several web platforms for assisting with primer design are available, such as www.ncbi.nlm.nih.gov/tools/primer-blast.
Once you have successfully acquired your DNA samples and designed your forward and reverse primers, you can proceed with the PCR experiment.
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Note: From the beginning of your PCR experiment until the end, you should always wear gloves in order to avoid DNA contamination. All the reagents, primers, and enzymes should be kept in ice. Make sure that primers, DNA template, and buffer are completely unfrozen before starting to prepare the PCR solution. It is important to create an experimental design in accordance with scientific guidelines, including a positive control and a negative control. As the negative control, you can prepare a PCR deprived of DNA template. For the positive control, you should use a set of primers and DNA template shown to work properly in previous experiments.
Depending on the DNA polymerase you use, final concentration of each reagent will vary. These are the standard volumes and concentrations taking into account a 50ul reaction:
Tip: Set up the PCR settings, such as temperature, time and cycles before your PCR reaction is ready. See the diagram below for reference of typical PCR reaction settings.
Note: Always pay attention to the guidelines of the DNA polymerase, since denaturation, annealing, and extension temperature and time highly depend on it. In the thermocycler, you should set up these steps subsequently (refer to diagram above):
These steps should be repeated for 25 to 35 rounds (cycles). A final step of extension is required to allow all the PCR products to be correctly synthesized, usually at 72°C for 10 min. Finally, the temperature should be reduced to 4°C to store the PCR product.
*Refer to PCR Principle Section (Data Acquisition and Analysis: Gel Electrophoresis) for detailed explanation of how to analyze PCR results*
Common DNA loading buffer (6X) recipe:
Note: In order to avoid staining with ethidium bromide, you can use Midori or Red Safe pre stained gels, which are less toxic compounds. Use a DNA marker to compare the correct size of the PCR product.
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Reverse transcription is the process by which RNA is transcribed into DNA, which will allow us to perform experiments such as qRT-PCR under the catalytic activity of a specific DNA polymerase that is only able to amplify fragments from DNA molecules.
Note: From the beginning of your PCR experiment until the end, you should always wear gloves in order to avoid DNA contamination. All the reagents, primers and enzymes should be kept in ice.
Similarly to standard PCR, a set of primers should be designed as described before in order to amplify each cDNA fragment of interest, usually corresponding to a specific gene. Moreover, it is also recommended to design primers specific for other cDNA genes for data normalization procedures, which are also known as “housekeeping” genes (e.g. 18S, GAPDH, ACTB).
Note: Set up the PCR settings, such as temperature, time, and cycles before your PCR reaction is ready.
For each cDNA gene, prepare the following reaction mix in 200 μl PCR tubes for a final volume of 50 μl:
When the PCR is finished, perform quantification analysis by comparative Ct method. The Ct method compares the Ct values of the samples with standards samples. The Ct values of the controls and the samples are normalized using the Ct values obtained for housekeeping genes. To validate the Ct calculation, it is required that the amplification efficiencies of the target should be close to endogenous. This can be accessed through the utilization of different template dilutions.
You can access more information about sample preparation, protocols, and troubleshooting in our PCR Technical Resource Center.