One of the problems in assays using 96 samples in a culture plate or PCR tubes and PCR tray is that it creates variability and increases the standard deviation. If the standard deviation of the samples is too high or the triplicate assay samples are very different from each other, that data point may be un reliable. One way to avoid this partly is to have ( 1) a solid platform to place the samples, ( 2) If the samples have to be kept cold for extended periods of time, keep them in a solid metal tube holder kept on ice. This will help all the samples to have uniform temperature and will work uniformly.
PCR has become a widely used tool in basic research as well as clinical diagnostics of a variety of infectious diseases. As the sample type gets complex, the technique also becomes complex, including sample prep for PCR. For the complex specimens, careful preparation of the samples is critical to achieve best results. How the samples are handled, how it is protected from degradation etc. For complex and sensitive samples any element of PCR, no matter how simple can affect the results with serous consequences, and costly waste of samples and reagents.
The steps before PCR, the sample prep is critical to the success of PCR. It is important to keep the reagents and samples for PCR cold to avoid thermal degradation. Specially samples of RNA, and Taq DNA polymerase enzyme. Keeping the reagents cold during PCR set up will protect the samples and reagents intact without any degradation, and will produce the most sensitive and consistent results. If the PCR set up is done without adequate care, the result can be misleading, and the experiment a waste of time. The following problems can arise if the reagents and samples are not kept cold throughout the preparation and partially degraded:
- High background for most of the samples
- Non- specific signal for the targets
- Low signal
For satisfactory results keep all the reagents and samples cold throughout the master mix preparation and sample addition
- Keep all PCR reagents on PCR Tube Rack (PCR Cool Rack), which is placed on ice after thawing.
- Keep the master mix on the PCR cool Block on ice
- Keep the reaction mixture in tubes on PCR cool Rack until sample addition.
- Prepare all the reaction mixture and add sample in a timely fashion for consistent results.
Designing of PCR Cool Block (PCR Cool Chamber)
Several years ago when I was working in laboratory, I did PCR very frequently. Each experiment had a lot of sample to process, mostly RNA samples extracted from cells or tissue. In those days we did the PCR using 0.2 ml individual tubes and 0.2 ml PCR tube strips. For setting up master mix for PCR and to keep the PCR reagents such as Taq Polymerase, and samples cold, I kept all the small 0.2 ml tubes on ice in a bucket. Keeping dozens of small clear tubes straight on ice was challenging, especially to add samples and keeping track of these tubes. After few months of this, it became very painful to go through the process and I knew there had to be a better way to do this efficiently. That is when I thought of designing PCR Cool Rack.
I went to a local machine shop and asked the owner to make a small tube holder (tube rack) to hold couple of dozen 0.2 ml, and 0.5 ml tubes. I started using them by placing it on ice in an ice bucket. The cool block was made of solid aluminum, and it kept the samples in the tube cold. No only I could keep the samples cold, it also provided a small stable platform to work with. I could prepare temperature sensitive samples in tubes on these cool racks.
During this time new PCR format started to emerge in the market. These were 96 well PCR plates, where you can do 96 samples at once. In order to accommodate the 96 well PCR plate I asked the machine shop to make a larger block with wells corresponding to the tubes. Since some of the reagents for the PCR prep came in 0.5 ml tubes and 2 ml tubes, which needed to be kept cold, made the PCR blocks with additional space to hold the tubes as well as a 96 well tray. After precision machining the PCR Block, the aluminum blocks are anodized to protect the surface from corrosion. Afterwards I made these and gave it to my colleagues to use. At this point I realized that if this item is useful for me, PCR Coo Block might benefit other researchers. I made this into a product and in the last ten plus years, it is very widely received in the market. After few years, I designed the Double PCR Cooling Block, which can accommodate two 96 well PCR tray and reaction tubes on the same block. It is very useful to process two plates or transfer samples from one plate to other. PCR cool block (PCR Cool chamber) also provide a stable platform to prepare large number of samples without worrying about keeping them straight, or floating in ice water when the ice melt. If you prepare 96 well PCR tubes or plates , tube strips, this is a very useful product.