Setting up a PCR lab from scratch

Setting up a PCR lab from scratch

AMPure XP by Beckman Coulter for PCR Purification

Setting up a new lab is exciting, but can also be a daunting process. We've recently faced this challenge ourselves, so we’ve put together our learnings in this four-part series. After discussing what is needed for nucleic acid extraction in part one, we'll now take a look at what needs to be considered when performing polymerase chain reactions (PCRs).

Table of contents
Infographic explaining the necessary steps to extract pure nucleic acids from samples

1    How do PCR, qPCR and RT-PCR work?

Real-time and reverse transcription PCR are both often abbreviated to ‘RT-PCR’ but they are not the same thing.1 To make sure that we're all on the same page, we will briefly outline the differences between these abbreviations – PCR, qPCR and RT-PCR – before exploring each technique further:

PCR

The polymerase chain reaction (PCR) is a fast and inexpensive technique used to amplify specific DNA segments. First, the sample is heated so the DNA denatures (separates into two single strands). The temperature is then lowered to allow oligonucleotides to anneal to specific segments of the single-stranded DNA, before being raised again to the optimum working temperature of the polymerase enzyme, which then makes a complimentary copy of the DNA. One repetition or ‘thermal cycle’ of these steps theoretically doubles the amount of the specific DNA present in the reaction. Depending on the amount of DNA present at the start, and the number of amplicon copies needed for post-PCR applications, 25 to 40 cycles are usually performed.

 

Interesting fact: The polymerase enzyme typically used in PCRs is called Taq-polymerase, because it was originally isolated from the bacterium Thermus aquaticus. First discovered in a hot spring in Yellowstone National Park in the 1960s, this enzyme can resist the high temperatures necessary for DNA denaturation.

qPCR

qPCR, also called real-time PCR, quantitative PCR or quantitative real-time PCR, is a technique used to detect and measure the amplification of target nucleic acids as they are being produced. In contrast to conventional PCRs, qPCRs require a fluorescently labelled oligonucleotide probe, and a thermocycler able to measure fluorescence and calculate the cycle threshold (CT) value. Typically, fluorescence increases in direct proportion to the concentration of the PCR product being formed, which allows target quantities to be measured in real-time.

RT-PCR

Reverse transcription PCR (RT-PCR) is used to amplify RNA target sequences, such as messenger RNA and viral RNA genomes. This type of PCR involves an initial incubation of the sample RNA with a reverse transcriptase enzyme and a DNA primer (sequence specific, oligo dT or random hexamer) before PCR amplification.

The abbreviation RT-PCR is sometimes also used for real-time PCR rather than reverse transcription PCR. We suggest that real-time PCR should be abbreviated as qPCR, since real-time PCR is also called quantitative PCR or quantitative real-time PCR.

 

PCR reactions are very sensitive and create a large number of copies of nucleic acids from minute amounts of starting material. This makes them a fundamental and highly effective molecular biology technique. However, because it is prone to amplicon and sample contamination, planning and designing of your PCR lab space will need careful consideration.

2    Designing your PCR lab

Ideally, a PCR lab should have two rooms with two areas, each designed for specific tasks. The first room should be exclusively used for pre-PCR activities and divided into a master mix preparation area and a sample preparation area. Air pressure should be slightly positive to prevent aerosols from flowing in.

The second room should have a dedicated area for nucleic acid amplification, and another one for product analysis. Air pressure should be slightly negative to ensure that amplicon aerosols don't leave the room.

If you're lacking in space or budget for a two-room PCR lab, you can set up the pre-PCR and amplification and analysis areas in the same room, but ensure they are as far from one another as possible.

Having pre-PCR activities spatially separated from the amplification and analysis area – either in different rooms or in separate benches – is very important, because you usually have a low amount of the nucleic acid sample during preparation and a very high concentration after amplification. This means that if you analyze your PCR in the same space as you prepare your master mix and samples, you may get false-positive results due to amplicon contamination.

You should also ensure that your lab set-up follows a unidirectional workflow. No materials or reagents used in the amplification and analysis areas should ever be taken into the pre-PCR space without a thorough decontamination. This means that you'll need dedicated equipment for each area, e.g., two different sets of pipettes. This unidirectional workflow should also apply to lab staff. If you've been working in the amplification and analysis areas, and you need to go back to the pre-PCR area, change your personal protective equipment, as it may have been contaminated by amplicon aerosols.

Another precautionary measure to take into account when setting up your PCR lab, in addition to the spatial separation, is temporal separation. You could, for example, consider setting up your PCR reactions in the morning, and perform the amplification and analysis steps in the afternoon. This may limit your flexibility, but will prevent contamination issues and having to repeat your experiment.

Clock explaining that the PCR set-up and the amplification and analysis shouldn't be performed at the same time

3    PCR equipment tips

PCR labs typically require a variety of equipment, such as centrifuges, vortex mixers, pipettes, fridges and freezers, thermal cyclers and analysis instruments (e.g., electrophoresis systems). Depending on the size of your lab and your applications, the amount of equipment you’ll need may vary. Instead of providing you a 'shopping list', we will outline what you should look for when purchasing equipment and consumables in order to keep contamination of your PCR reactions to a minimum.

3.1    Laminar flow or biosafety cabinet

Since you can never be 100 % certain that there are no amplicon aerosols in your pre-PCR space, you should set up your PCR reactions in a laminar flow hood or biosafety cabinet, decontaminated with a bleach solution prior to starting and after you finish your work.

3.2    Pipette tips and other consumables

Despite being more expensive than normal pipette tips, using filter tips for your PCR set-up will avoid aerosols entering and contaminating your pipette, and avoid aerosols that might already be present in your pipette contaminating your master mix or samples. To minimize your filter tip consumption, first fill all your tubes with the master mix using only one tip or set of tips – if you're using multichannel pipettes – and follow with your samples, using one tip per sample. Adding the sample last is also recommended because it's easier to dispense it into a liquid than into an empty tube, and because it reduces the risk of aerosolizing your sample as you pipette. Learn the best pipetting practices to help prevent aerosol formation.

For consumables, you should make sure that you have enough small vials available in your lab when your PCR reagents arrive. Aliquoting them into smaller containers will increase their shelf life and prevent them from going through too many freeze/thaw rounds. If your reagents get contaminated, it will also save you from throwing away your entire supply, as you’ll have clean aliquots available for a second PCR.

Finally, you’ll need to make sure that all consumables and equipment are free of DNase, RNase and PCR inhibitors. Always chose sterile products from manufacturers that can certify that their tips and consumables are free of any of these potential contaminants.

pipette tips certified free of DNAse, RNase and PCR inhibitors

4    Cleaning and contamination control

You won’t need to worry about cleaning or contamination control when setting up your lab, but you will when your lab is up and running. We will briefly address this topic below.

Whether you decide to set up your PCR reactions in a laminar flow hood, a biosafety cabinet or an open bench, you will need to decontaminate your work space before and after set-up by wiping it with a freshly made bleach solution and distilled water. The same process should be performed in the amplification and analysis areas. You should also make sure you clean your pipettes, equipment, doorknobs, and the handles of your fridges and freezers regularly.

Because PCR assays are so sensitive, all the preventative measures described here may still not guarantee that your experiments will never get contaminated. It is therefore necessary to include the appropriate controls to detect contamination early. Always include negative and positive controls, as this will help identifying master mix contaminations, and confirm the performance of the extraction protocol, reagents and amplification steps. Additionally, you should monitor the positivity rate in your lab, and ensure that unexpected increases in detection have identifiable causes, e.g., a seasonal outbreak.

5    Conclusions

We hope that you are still just as excited about setting up your PCR lab, and that this blog has made the task less daunting for you. If you're curious to what you'll need for subsequent nucleic acid quantification workflows, subscribe to our newsletter to get the third part of our molecular biology lab set-up series directly to your inbox once it's available.

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