96 well plate DNA extraction
DNA extraction from 96 well plates is commonly used in molecular biology and biochemistry labs. This process can be complex and time-consuming, but it is essential to many scientific experiments.
This article will discuss the basics of 96 well plate DNA extraction and provide some tips and tricks to help you successfully extract DNA from 96 well plates.
Collect the Necessary Materials
Before beginning the process of extracting DNA from a 96-well plate, you must gather all the necessary materials. Depending on your method of choice, you may require different equipment and solutions.
The basic materials needed for a 96-well plate DNA extraction include a 96 well plate template, lysis buffer, proteinase K solution, ethanol, centrifuge tubes, micropipettes and pipette tips, a centrifuge, filter paper, and collection tubes. You may also need other solutions and materials specific to your chosen method.
Prepare the Samples
Before beginning the DNA extraction process, you must prepare the samples. This involves ensuring that your samples are labeled and arranged in the 96-well plate.
Place the samples into the appropriate wells and leave enough room in each well for the reagents. To avoid cross-contamination between samples, ensure that you are working cleanly and that no pipette tips have been used between samples. Once the samples are properly arranged in the plate, you can begin the extraction process.
Add the Lysis Buffer
The next step in the 96 well plate DNA extraction is to add the lysis buffer. The lysis buffer comprises detergents, salts, and enzymes that break down the cells.
Before adding the lysis buffer, create a 96-well plate. This will make it easier to track which wells have the lysis buffer and which don’t. To create a 96-well plate, use a 96 well plate with labels and a marking pen.
Once the template is completed, add the lysis buffer to each well of the 96-well plate. Make sure to carefully read the instructions for the lysis buffer for the correct amounts of buffer for each sample.
Once all the lysis buffers have been added, seal the plate with parafilm and mix by inverting it 5-6 times.
Add the Proteinase K
The next step in the 96 well plate DNA extraction process is to add the Proteinase K. This enzyme helps to break down proteins and other components of cells, helping to release the DNA from its surrounding components.
To do this, prepare the Proteinase K by diluting it in a suitable buffer, according to the manufacturer’s instructions.
Then, add the diluted Proteinase K to each of the wells in the 96-well plate. Ensure that each well contains an equal amount of Proteinase K, and be careful not to spill any of the solutions out of the wells.
Once all the wells have been filled with the Proteinase K, incubate the plate for an appropriate length, typically about 1 hour. After incubation, move on to the next step of the DNA extraction process.
Incubate the Samples
Once the lysis buffer and proteinase K have been added to the samples, the 96-well plates should be incubated appropriately.
The incubation time will depend on the sample type and the components’ concentrations. During the incubation process, the contents in each well of the 96-well plate will be digested by proteinase K, releasing the DNA from the cells.
The optimal temperature for this incubation process is between 50 and 60 degrees Celsius. After incubation, the next step is to add ethanol to the wells and centrifuge the samples.
Add the Ethanol
Once the proteinase K has been added and incubated, it is time to add the ethanol. Begin by labeling a new 96-well plate with your sample names or numbers.
Pipette 500μL of absolute ethanol into each well of the template, making sure not to mix up any samples.
The absolute ethanol will help precipitate the proteins and create a DNA pellet. After preparing the samples, gently rock the plate for 1-2 minutes to ensure thorough mixing.
Centrifuge the Samples
Centrifugation is an important step in the DNA extraction process. To centrifuge your 96 well plates, place them in a centrifuge tube rack and insert them into the centrifuge.
Then, set the centrifuge to the correct speed and time. The samples should be centrifuged for 5 minutes at 13,000 RPMs.
This will allow the lysate to settle into layers in the bottom of the wells, allowing for easier removal of the proteins.
Wash the Samples
After centrifuging the samples, you must wash the 96-well plate with a buffered solution. This is done to remove any remaining impurities and to help increase the purity of the DNA extracted.
To do this, carefully remove the plate from the centrifuge and add 500 μL of either TTE buffer or a washing buffer to each well.
Once again, using a pipette, mix gently and thoroughly by pipetting up and down 8-10 times. Centrifuge the plate at 5,000 RPM for 1 minute and carefully discard the supernatant.
Repeat this step with a second 500 μL aliquot of either TTE or washing buffer. After the second centrifugation, discard the supernatant and allow the plate to air dry for 10 minutes.
Elute the DNA
Once the DNA has been successfully washed, eluting it from the 96-well plate is time. To do this, you need to add a low-salt buffer solution to each well.
This will help loosen the bonds between the DNA and the silica particles so they can be released. After adding the buffer, mix the contents of each well using a pipette, then incubate the plate for 3-5 minutes at room temperature.
Once the incubation period is finished, centrifuge the plate at 2000 rpm for 2 minutes. This will help separate the DNA from the other components of the sample.
After centrifuging, discard the supernatant and add fresh low-salt buffer to each well. This will help elute the DNA from the template.
Finally, collect the eluted DNA by transferring it to a new tube or plate. Store it at -20°C until you’re ready to use it.
Store the DNA
Once the DNA has been eluted, it is important to store it correctly so that the quality of the sample is not compromised.
For a 96-well plate, it is best to store the sample in the same plate in which the extraction was performed and seal the plate with a sealing film or tape.
The plate can then be stored at -20 °C in a freezer. If it is necessary to transfer the samples to another container, this should be done using a micropipette and PCR-grade tips to avoid cross-contamination.
It is important to label the containers clearly with the sample ID, date, and concentration. Once the samples have been stored, they can be used for downstream molecular applications such as PCR, qPCR, and sequencing.
DNA extraction from a 96-well plate is a simple, efficient, and reliable way to acquire large amounts of DNA from various samples.
Following the step-by-step procedure, researchers can obtain high-quality, pure DNA for further analysis.
This method also allows for easy scaling of the extraction process; since it is conducted in a 96-well plate, researchers can perform multiple extractions simultaneously. As a result, this technique saves time and resources for any laboratory undertaking such analyses.
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