PCR (Polymerase Chain Reaction) is a ground-breaking technique, which was developed by Kary Mullis during the 1980s. PCR is depended on the ability of DNA polymerase to develop new DNA strand complimentary to the template strand formed.
PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. As DNA polymerase can add a nucleotide only onto a pre-existing 3'-OH group, it requires a primer onto which the first nucleotide could be added. This requirement marks it clear to define a typical region of template sequence that the researcher wants to amplify. Read this article further to learn what are the pre-PCR and post-PCR prerequisites of a polymerised chain reaction.
Pre-PCR space is any area that is used to prepare components of a PCR reaction. The key points for pre-PCR set-up are: Minimize the size of reagent aliquots as much as possible, keep consumables away until absolutely needed, and keep template as isolated as possible.
PCR Template Issues
The common PCR template issues are: Matrix issues (never assume that a template is pure of its matrix e.g. blood, tissue, etc.), identification issues, PCR issues may be specific to sample conditions, cross-contamination issues. Keep samples separate at all costs.
Templates in pre-PCR Areas
Keep templates away from pre-PCR areas until absolutely needed, If possible aliquot templates. This allows for re-testing in case of any issues. All templates must be properly located before loading. Need to make sure the right template is in the right location.
Any hood where PCR prep work is performed must have positive pressure. This means that the air pressure within the hood leads to air flowing out. Any air filters must be subject to regular maintenance.
Post-PCR Room is the room where the actual reactions take place. Everything should be well-sealed. All reactions should be logged. Keeping track of what PCRs were performed often helps mitigating problems.
Post PCR Room Maintenance
Post PCR maintenance tips include, bleaching out PCR cycler area regularly, fool proofing reactions from leaking, contaminating surfaces, and stopping, avoiding use of PCR cyclers for short-term storage whenever possible. The less unnecessary contact PCR cycler’s product has, the better it is.
Best Practices in PCR Assays
The best practices in dealing with PCR assays are: Finding the simplest means of solving a problem. Fresh reagents can solve many quality problems, and gels can detect much more. Understand your assay. Some problems may be due to a product of assay design. Always think of potential pitfalls.