real time PCR template - template concentration (Aug/16/2007 )

My qPCR standard curve has not been working on DNA concentrations over 20ng. I'm using the same exact tube of template in different concentrations but the higher end does not work. I'm aware that high template concentrations can inhibit qPCR reactions but my max is only 40ng right now, this does not seem unreasonable. Please advise.

Also my triplicate reactions are not quite perfect (some are far from perfect), any suggestions?

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Hi,

1.Maybe you should check back your primer efficiency.try run gel electrophoresis and see whether the pcr product really exist.
2.This may sound weird, but why don't you try with higher concentration of dna? (80-100ng).Mine work perfectly at 110ng.
3.Regarding the triplicates,I also had the same problem when first starting with real time.I think it is due to some 'micro' technical error(pipetting).try to load your template first then add the pcr mixture.

Anyway, these are just some suggestion.Good luck and have fun!

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As far as getting your curves to be reproducible, you need to make sure you are working with a very good pipet. If you are adding 1 microliter of template, and using a pipet that is perhaps mis-calibrated or whose O-rings are getting old or some other problem, then the basis of your problem might be that you are not actually pipetting exactly a microliter when you think you are. Some liquid adheres to the outside of the tip, as well, which can mess up things. We use a very good pipetman for adding template, which is only used for QPCR and nothing else. I calibrate it before each experiment as well - By pipetting one microliter of water onto our analytical balance (should get a reading of 0.0010 g when you pipet a microliter of water.)

As far as the concentration: It isn't clear how much you are actually adding, or what the source of template is. A concentration, by definition, is an amount per volume. When you say 20 ng, is that per microliter of reaction? I assume that what you mean is that you are adding twenty ng into a reaction tube. What is the source of template DNA? If you want to avoid inhibitory substances, you should consider CsCl banding your template DNA. And, is your template DNA from a plasmid, or is it a genomic sample?

I make my standard curves from CsCl cleaned, linearised plasmid DNA that has my target gene on it. The mass (ng) that I add is less useful information than the corresponding copy number that I am adding. For example, if I add 20 ng of plasmid DNA, I am adding about 10^9 copies of my target. but if I add 20 ng of genomic DNA, I will only be adding maybe 10^6 copies, because the target is so much rarer, relatively speaking, in genomic DNA than on a small plasmid.

I suspect your DNA needs to be CsCl cleaned. We routinely run into problems with inhibitory substances in our DNA stocks unless they have been banded in a gradient.

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Thanks. I think you're right. I need to further purify my DNA. I did another experiment today where hardly anything worked on my gDNA standard curve but the same primers worked fine on another set of samples that had been PCR product purified. So it's obviously some contaminants left over from my prep. It's so obvious that I completely overlooked this possibility. Thanks for taking the time to read and answer my post. I'm sure I'll be asking some more stupid questions soon!

As far as the pipetting goes, you're probably right again. Unfortunatly we don't have a pipet that we can dedicate for qPCR only but I plan on making a serial dilution standard curve next time that allows me to pipet in higher volumes than 1ul. The good news is that data that came out looks really good. Thus with a new and perfect gDNA standard I will have perfect data and publish a wonderful error free paper:o)
Thanks again!

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