In this article, I will explain how to easily prepare PCR stock primers and how to dilute them into a working primer solution.
Mastering qPCR
A video tutorial on how to dilute new qPCR primers can be found in our Mastering qPCR course
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Before you start
So you have designed and ordered your primers and now they have finally arrived. But how do you prepare lyophilised (freeze-dried powder form) oligo primers for use in PCR? If you are unable to prepare the primers straight away, then store them at 4oC until the time comes.
Before you start, here are some general tips when working with PCR primers:
- Make sure you work in a DNA-free environment. Preferably in a PCR preparation hood. This is to avoid contamination of your stock and working primer solutions.
- Use PCR-grade water (DNase- and RNase-free) to reconstitute and dilute your primers.
- Use filter pipette tips to prevent contamination via pipetting.
To prepare primers for PCR, just follow these two simple steps:
1. Reconstitute your stock primers
First things first, you should briefly (approximately 30 seconds) centrifuge your primers to pull all of the lyophilised powder to the bottom of the tube. Otherwise, if the powder is stuck in the lid while opening the tube, you could potentially damage your primers.
Next, you need to reconstitute your primers with PCR-grade water to make a 100 μM stock solution. But how much water do you need to add? The amount of PCR-grade water to add depends on the number of nanomoles (nmoles) for that oligo primer. This can usually be found on the tube itself or the primer sheet supplied with the order.
For every 1 nmoles, add 10 μL of PCR-grade water.
For example, if a primer states 19.4 nmoles, then add 194 μL of PCR-grade water.
Mix the solution by vortexing to reconstitute the primers. Store primer stocks at -20oC.
2. Make a working primer solution
You should never use the stock primers directly into a PCR because they are so concentrated. Working from one tube is also a bad idea. It only takes a bit of contamination to creep in to the tube and you will have to re-order the primers again.
Therefore, it is best practise to create working solutions that are of lower concentrations. The concentration of choice for the working primer solution is totally user-determined. The most common concentration for a working primer solution is 10 μM.
To make a 10 μM working primer solution, follow these steps:
- Add 10 μL of primer stock solution to an RNase- and DNAse-free tube.
- Add 90 μL of PCR-grade water.
- Mix by vortexing.
Aliquot and store working primer solutions at -20oC. Avoid excessive freeze-thawing of working primers.
Featured image credit: MadLab Manchester Digital Laboratory (via Flickr)
