On Thu, May 23, 2013 at 6:03 AM, Jeswin <phillyj101@gmail.com> wrote:
-- > also say you want to know if there is horse meat in your food, or dolphin> meat, or listeria, or E.Coli O157:H7? Again qPCR is the gold standard.I thought about that sort of stuff, on the DIYbio level. But what
makes qPCR a better alternative than regular PCR and running a gel?
Getting cheap qPCR enzymes that also give good signals are difficult.
You can't use regular Taq.
Well for something like GMO detection, you probably want to quantify how much there is. For something like Listeria, maybe you don't care exactly how much there is, but only want to know if there is any at all. Then yes you could do endpoint PCR and run a gel. If you already had a PCR machine and gel apparatus this might be the easiest way to run one sample. But there are some advantages to the qPCR:
- If you already have a qPCR machine, I think qPCR will be cheaper than casting and running a gel, and will *certainly* save a lot of time. There are cheaper enzymes out there, and also dyes like EvaGreeen.
- If you are doing any sort of real testing with lots of samples, you may not want to open the tubes after PCR. During the course of related testing for this machine, I ran 100's of PCR reactions and was doing many things with amplicon products like putting them in fluorometers and running gels to compare. I was not overly careful about this and now my negative controls often amplify for this assay, even with different pipettes. And I did check that it is amplicon and not phage contamination as when I switched the primer set to another region, the negative controls were ok again.
One other way to do this cheaply in a DIYbio context: prepare your PCR reaction, and then aliquot it into 2 tubes. Run only one in PCR. Then add a cheap dye like GelGreen to each, and compare the tubes in a transilluminator, and look for an obvious increase in fluorescence in the PCR tube. Of course you won't know it's the correct amplicon, but this is an option.
Colony screen them? In all of the cloning I have done, if the screen
> But I'm even interested in the most mundane uses. Say you have ran a PCR
> reaction, and now want to send it off for sequencing to do DNA barcoding. Or
> you PCRed something to clone. But you don't know if the PCR worked. Why
is positive, then I am ~95% sure the sequence is fine. There was just
1 or 2 cases where there was an insignificant mutation outside the
region of interest.
My point was to not even colony screen them unless you knew the amplification was good. This is more of an engineering point that I don't think is very commonly held by biologists right now, but should be. Basically I find myself often doing one step to verify a previous step, or doing a multi step process with no intermediate verification, and then realizing a failure occurred only at a later step. So I not only wasted time in doing later steps, but have to spend more time finding where the failure occurred. If verification happened at each step, especially if it happened cheaply, transparently, and automatically, that would be a big win for engineering biological systems.
I first did qPCR because I wanted to measure gene expression, but then after seeing this, I never wanted to go back to endpoint PCR, because of this benefit. Once you have the machine it is cheap, so I see no reason not to do it, unless you have a specific case where fluorophores will not be cleaned up and will cause a problem.
-Josh
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