PCR’s best quality is its exquisite sensitivity. One can generate over 10 million copies of target DNA or RNA sequences in as little as 32 cycles using short oligonucleotides or primers. PCR’s sensitive nature permits scientists to be abstract and amplifies DNA from raw samples to get a beneficial DNA profile.
This level of sensitivity particularly applies in a laboratory. In contrast, if not taken care of, it can also create issues to avoid contamination with other templates and amplicons that may be available in a laboratory. These contamination problems can result in the wrong template amplified, i.e., false positive results (a test result that incorrectly shows a particular condition or attribute exists).
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Laboratory Construction
Laboratory contamination prevention begins with the construction and distribution of the areas of a PCR laboratory. At the very least, two areas should be designed for PCR testing, and that is pre-PCR and post-PCR.
The pre-PCR area should contain two further divisions: sample preparation or addition to the master mix and PCR master mix preparation.
- Using a Dead Air Box (DAB) with UV light, master mix preparation can be separated from sample addition. For master mix preparation, the DAB houses consumables, and small lab tools (PCR Tubes with Flat Caps, vortex, mini-centrifuge, tips, pipettors) are required.
- Sample preparation contains a manual or automated extraction. Consumables and small equipment (mini-pipettors, centrifuge, vortex, treated plates, tubes, tips, etc.) required for sample preparation could either be on a Biosafety Cabinet (BSC) or bench top, in another DAB, depending on the samples type. Moreover, this area is also used to add samples to the PCR master mix.
- A substantial refrigerator/freezer is required to keep the kit and another storage of samples frigid.
The second room or post-PCR should be created for post-amplification and analysis. Here the thermal cyclers performed tasks related to PCR amplification. A lab worker will perform all the steps for manipulating open tubes after PCR amplification in this area. This room should also require house consumables and small tools (vortex, mini-centrifuge, tubes, pipettors, and tips) for post-PCR analyses and preparation.
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Environmental Control
Ensure that independent environmental control is given to both areas (Post-PCR and Pre-PCR). Keep away both areas from having ductwork for air conditioning. It additionally equipped both rooms with air-lock doors.
Remember to make all efforts to set up work areas as far apart as possible when separate rooms cannot be established for Post-PCR and Pre-PCR work. Lab technicians must work in such a way for these two areas as working in separate rooms. Wearing different personal protective equipment (PPE) in each region is vital for the care of the laboratory.
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Unidirectional Workflow
Keep the molecular lab workflow in a single direction, as pre-PCR is greater than post-PCR. The pre-PCR room is only used to prepare only PCR master mix reagents and samples that are PCR templates.
In the post-PCR room, some tubes have undergone amplification containing amplicons (amplified template). And therefore, it should never open in the pre-PCR room in any situation. Amplicons templates can be used for future PCR reactions and contaminate sample preparation reagents, equipment, consumables, or PCR.
Thus, the post-PCR consumables and PPCE (gloves, lab coats, goggles, etc.) should not be placed back or used in the pre-PCR room without intensive decontamination. A lab technician must change PPE when moving from room to room. If possible, technologists who performed post-PCR tasks should avoid working in Pre-PCR. If any laboratory worker goes against the unidirectional workflow, then change PPE.
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Pipetting Technique
The proper pipetting technique is essential to get the best performance and quality of your result in any molecular assay. The appropriate pipetting process helps to reduce contamination between samples and otherwise generates a false positive impact.
The correct pipetting method confirms the exact volume is dispensed and aspirated. This technique also evades splashing during liquid dispensing. To avoid any sample splashing, open and close all tubes of samples and reaction plates cautiously.
Conclusions
We’ve covered four essential methods to remove contaminants. Following these methods stops contamination from ever becoming an issue in a laboratory.
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