I use Bio-Rad's SYBR Green Master Mix for my qPCR work which, according to the manufacturer's instructions calls for a 50uL reaction volume. I use 20uL on a regular basis without any problems. As I am running a lot of qPCR in the future and looking for ways to save some money, I was wondering if it's realistically possible to use a 10uL reaction volume and achieve the same results in terms of well-to-well variability. My main concern is the inevitable increase in pipet ting error as the reaction volume decreases.
So, does anyone use 10uL qPCR reaction volumes? Have you ever had any issues?
I did frequently set up 10uL-qPCR tests using the master mixes of Roche Diagnostics only when I was not concerned about the sensitivity due to low DNA concentrations; no problem.
I did frequently set up 10uL-qPCR tests using the master mixes of Roche Diagnostics only when I was not concerned about the sensitivity due to low DNA concentrations; no problem.
I've used 15 uL-size reactions with LCM-qPCR samples and got stable/reliable results. Per each 15 uL rxn, 3.6 uL was the sample contribution to total volume. Pioneer/Dupont runs 8 uL-size rxns. Automated however. I would give it a shot with 10 uL-size reactions and not fear anything but pipetting itself. Be sure to weigh water and remember that 0.99754 g of water = 1.0000 mL of water at 23C (~room temp) to calibrate every pipet usage. Even freshly 'calibrated' pipets can lie, so weighing water with every pipet adjustment is a tangible way out of that caveat.
Hi, I am doing 10µl qRTPCR reactions using Promega qRTPCR kit which is also suitable for fast PCR and it is working very well.
In Roche plataforms for qPCR 10µL can be conducted fine. Good repoducibility but like Wafa said sensitivity for low abundance targets ,ay be afected.
We chose 10ul to do the qPCR not only to save the cost but also with good results, and you can try bio-rad iq SYBR Green regent or taqman regent from life corpration.
Hi there. I do it every day and it works great. I have done it in several PCR machines (Stratagene 3005, BioRad CFX96 and 384). However, if you want to be sure, my advise is to run aPCR with both volumes (10 and 20 µl) and compre.
Good luck with your experiments
Hi Edward,
You can run 10 ul reactions but as Francisco mentioned, I would run both 10 and 20ul reactions and compare your results (ideally comparing standard curves result). The major difference between 10 and 20 ul is that a smaller volume will show you more easily errors in your pipetting than a larger volume would. You need good pipetting technique and calibrated pipettes.
Best
10ul or 20ul reaction volume does not matter until you maintain the required conc. of primers, so according to my experience both have been working with same results.
I usually perform qPCR in 96 well in 15-20 ul and in 5-10 ul on 384 wells on applied biosystems instruments and it works very good
Hi Edward,
I only run 10µl qPCR reactions and have had no issues in the past. but to reiterate Nandhini, you must correctly maintain the reaction conc. ratios.
All the time, no problems. Pipette error can actually be minimized. For example, using a 20-200ul pipette for a 25ul reaction vs. a 2-20ul for a 10ul reaction. Dilute your template and use 2 ul template in 8 ul reaction mix.
Routinely...I use 10 uL final volume for all my PCR reactions, unless I need a larger amounts of PCR product for downstream applications.
I've been using 10 uL reaction volumes for both regular PCR as well as qPCR without any problems for over 3 years now. Variability between samples (I run them in triplicate most of the time) is negligible in my experience. Pipetting error is always a concern so I would highly recommend a very good pipettor that has been properly calibrated. I use a Thermo Scientific Matrix automatic pipettor and have achieved highly reliable and repeatable volumes for any PCR or qPCR reactions that I do.
When going from 20uL to 10uL, do you just lessen the volume of mastermix?
Hello,
I always used to run qPCR in 10ul total volume. Using 4ul of cDNA allowed me to handle pretty well pipetting errors. Good luck!
Cody, you lessen the volume of everything but maintain the same ratio. So if Master mix goes from 20 to 10 then primer goes from 10 to 5 and template goes from 10 to 5. This example is just for your reference.
Hi, a quick question? I just shifted to 10ul from our regular 25ul PCR protocol. I used SYBR Green in BioRad CFX. During set up the program, I forgot change the reaction volume from 25 to 10? I have already done more than one plate which I will compare each other. Is that matters in my comparison?
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