Best pipetting practices in qPCR: Watch on demand

Quantitative PCR (qPCR) has emerged as one of the most popular techniques used for the quantification of nucleic acid molecules. This standard laboratory technique is based on the polymerase chain reaction (PCR) and is frequently used for gene expression analysis by quantitating changes in gene expression from biological and environmental samples. Despite qPCR now being a well-established analytical method, many limiting factors can influence qPCR effectiveness, including pipetting technique.

In this on-demand SelectScience webinar, Paulus Artimo, product manager at Sartorius Pipetting and Dispensing, shares best pipetting practices to help you master your pipetting technique and achieve greater efficiency in your lab.

Read on for highlights from the live Q&A session or watch the full webinar on demand.

Q: I know that Sartorius offers the Pipette Safe-Cone Filters can these be used instead of filter tips for contamination prevention?

PA: The Pipette Safe-Cone Filters that are inserted in the pipette nose cone shouldn't be used as an alternative to filter tips because the filter tips protect the sample from cross-contamination. The Safe-Cone filters only protect the insides of the pipette but not the outside of the nose cone and in order to be as effective, they would have to be changed after every pipetting cycle.

Q: Should I always keep my PCR agents on ice when doing sample preparation?

PA: This is a common practice in PCR labs. We should really refer to the kit manufacturer's instructions or your lab's standard procedures but remember that when pipetting colder- than- room- temperature liquids, you get more accurate results by not pre-reading the pipette tip. Some pipettes also offer user adjustment that can be used to account for liquid properties, reducing the accuracy, in this case, cold liquids. Please refer to the user manual of your pipette if you want to know more about how the user adjustment works or if it's available for you.

Q: What is reverse pipetting and can you use it all the time?

PA: Reverse pipetting is when you aspirate. First, you press the plunger all the way down to the second stop, you aspirate your target amount of liquid, and then a little bit extra. Now, you only then dispense until the first stop and then remove the tape from the vessel, and this way you get the target amount there. Reverse pipetting can be used with all liquids, but for aqueous liquids, forward pipetting, or so-called normal pipetting is as accurate when performed correctly. It's a bit faster, and of course, a smaller amount of liquid.

Q: What would your advice be for those working in a small lab and would you suggest that they try to separate these processes?

PA: Taking some of the principles of the unilateral flow or the unidirectional flow, meaning that when you start with the reagents, you never bring anything back from sample preparation to any other working area. If you were able to separate these working areas, even in a smaller lab, that would be great. If you have these different areas, then have dedicated pipettes or at least pipette tips, and try to hold all containers closed for as long as possible so you don't get any aerosols from the room air.

Q: Can you put pipettes in an autoclave to sterilize them and what would be best between alcohol and autoclaving for complete sterilization?

PA: For sterilization purposes, autoclaving is the best way. By sterilization, meaning that there are no microbes living on it. Ethanol or alcohol is good for wiping and overall cleaning, but for complete sterilization, autoclaving the pipette is the best way. Check that your pipette is autoclavable because otherwise you will just ruin the instrument.

Q: What is the best way to remove amplicons?

PA: Here we refer to any nucleic acids, I would suggest using commercial nucleic acid- removing agents like wipes or liquids. Ethanol is not appropriate as mentioned, as that's how you store nucleic acids. Go for some DNA AWAY or DNA Zap and other commercial removing agents.

Watch this webinar to find out how to master your pipetting technique>>