PCR (polymerase chain reaction) is a powerful tool for replicating and amplifying DNA sequences and is widely used in many fields, from medical diagnostics to forensics. A 96 well plate is an essential piece of equipment to conduct a PCR. Preparing a 96 well plate correctly is key to achieving optimal PCR results. In this blog post, we will provide step-by-step instructions on how to prepare a 96 well plate for PCR.
Place the 96-well plate in the laminar flow hood.
When working with any biological materials, it is essential to maintain a sterile environment. The laminar flow hood is designed to keep the work surface free of contaminants. Begin by placing the 96-well plate onto the laminar flow hood. Make sure the plate is stable and cannot be disturbed while working. Put on gloves to protect yourself from any contamination.
Use a sterile technique when handling reagents and samples
When working with a 96 well plate for PCR, it is important to use a sterile technique. The sterile technique involves wearing gloves, using sterilized tools, and working in a laminar flow hood. This prevents contamination of the samples and reagents used in the PCR reactions. Additionally, when adding samples to the wells of the 96 well plate, it is important to use a 96 well plate template to ensure that each sample is placed in the correct well. This helps to prevent cross-contamination between different samples and ensures accurate results.
Add reagents to the wells.
Before adding reagents, it is important to have a 96 well plate to ensure the correct amounts are added to each well. The amount and type of reagents may vary depending on the specific PCR application. Common reagents include primers, polymerase enzymes, buffer solutions, and dNTPs. When pipetting the reagents, keep the pipette tip close to the surface of the liquid to avoid bubble formation. Pipette slowly, as too much force can cause cross-contamination between wells. Once all the reagents have been added, use a paper towel to dab the edges of the plate gently. This will help keep the reagents from spilling out when the plate is sealed.
Add samples to the wells.
To add samples to the wells of the 96-well plate, first, prepare a 96 well plate. This template should indicate which well contains which sample. Before adding samples, label the plate on the side with a permanent marker so that you can keep track of the samples added. Once the template is prepared, use a multichannel pipette to draw up the sample and dispense it into the corresponding well on the plate. When all samples have been added, ensure each sample is in its designated well by checking the template and the labeling on the plate.
Seal the plate with parafilm.
Once you have added the reagents and samples to the 96-well plate, you must seal the plate with parafilm. Parafilm is a waxed paper-like material used to create an airtight seal around the wells of a 96 well plate. To seal the plate:
- Place a small parafilm over each well.
- Ensure that the parafilm covers the top of the plate to ensure that no air gets into the wells.
- If you are using a 96-well plate with additional features such as an external lid, ensure that this is also firmly closed before sealing with parafilm.
Incubate the plate.
Once all the reagents and samples have been added to the 96 well plate, the plate needs to be incubated. This is usually done in a thermal cycler or PCR machine, which can maintain a consistent temperature for the duration of the experiment. Depending on the experiment, the plate may need to be incubated anywhere from 30 seconds to several hours. Please read the instructions carefully and follow them precisely to ensure accurate results.
Analyze the results.
Once the 96 well plates have been incubated, you can begin to analyze the results. Visual inspection of the plate can help determine which wells contain PCR products. Additionally, a variety of plate readers can be used to analyze PCR products and quantify the amount of product produced from each well. These plate readers require a 96 well plate to read and interpret the data. In some cases, the software is available to assist in interpreting the results.
Preparing a 96 well plate for PCR is a relatively straightforward process. However, it requires meticulous attention to detail to ensure the accuracy of results. A 96 well plate can help organize the wells and samples and make the process easier. Be sure to take the necessary precautions when handling reagents and samples, and seal the plate securely with parafilm to prevent cross-contamination. You can obtain accurate PCR results from your 96 well plates with proper preparation and technique.