The term real time PCR essentially refers to visualising the amplification of exact DNA fragments as they are happening now, in other words in real-time. In being able to analyse the amplification of DNA fragments as they are occurring in real time, it enables scientists to quantify exactly how much DNA was in an original sample and ultimately deliver much more information about DNA than standard end-point PCR.
There are certain methods and tips that you can apply in order to optimise the effects of real time PCR.
1- Protect primers stability by storing them in a buffer
Instead of placing primers in water, which could potentially damage the DNA over time, particularly if the pH in the water is low, place the primers in a buffered solution, which is PH neutral, as this will protect the buffers from acid hydrolysis, which can damage DNA.
Putting 1 mM of EDTA in the master stock is also a good idea, as this will help protect against DNases occurring as you dilute the primers for working stock.
2- Avoid excessive freezing and then thawing and contamination and aliquot the primers
When you have a master stock, which is typically between 100 – 200 mM , it is likely that you will then want to make up stocks that you are not constantly having to freeze and then thaw for your primary source.
It is advisable that you prepare between 10 and 20 mM of working stocks in a buffer that is pH neutral and prepare aliquots that enable you to freeze and then thaw the working stock approximately three times or five at the most.
It is in your interest to limit the amount of times that you are freezing and thawing the primers, as doing so continuously can lead to some breaking down occurring in the primers, which will ultimately result in less efficient PCR and sensitivity.
In preparing aliquots you will significantly help avoid problems associated with the contamination of primers.
3- Mix the reagents thoroughly before use.
When carrying out real time PCR, reagents required to be mixed in the amplification reaction. These reagents contain enzymes, nucleotides and dyes, that are likely to have become separated and settled when they were stored in a freezer or refrigerator.
It is therefore important that you mix the liquid thoroughly to ensure that the dye, enzymes and nucleotides are variegated as this will avoid uneven distribution of reagents between the samples.
Writer Joyce Springs wrote this blog on behalf of www.primerdesign.co.uk.