I'm considering performing a PCR on my gene sets (both GOIs and house keeping genes), run the PCR products on a gel, extract them from the gel, and then measure their concentrations (using a nanodrop).
By knowing the exact concentration of that PCR products, would it be valid to use them as standards for qPCR positive controls, and for which to form standard curves?
It seems quite straight forward. This is my first time doing qPCR, so I'm wondering if this is actually the normal method of forming positive controls and standards.
Hello Martin,
theoretically, yes, why not, even though nanodrop is not very accurate and is affected by many parameters.
Why do you need to seperate your pcr products in a gel? When you run each positive control with your specific primer pair seperately you don´t need the gel. Clean up your pcr product and measure the concentration.
That should be fine.
I do this routinely, Martin, so I would say: 'yes, it's fine'.
Assuming your gel extractions are clean and high-quality (good 260/230 ratios are the real test), the purified, quantified products should serve as entirely adequate for generating standard curves. A quick test would be to titrate them down in series from say, 10^9 molecules per ul to 10^-1 per ul or so: you should see a very nice linear increase in Cq value that becomes progressively more inconsistent as you approach very small numbers of molecules, and that (if extrapolated) crosses the axis at a Cq of 34-37 or so.
As Dirk notes, you don't need to do this for a positive control (where 'product: yes/no' is the question), only for standard curves (where number of molecules is a key variable). Gel extraction is essential for the standard curve, though, as primers, primer dimers/unincorporated nucleotides in the PCR reaction will affect nanodrop reading.
Most folks will clone their gene-of-interest and use that cloned DNA to create their standard curve for their PCR. It's more time up front to clone, transform, grow up cells, etc, but it's easier to get a useful concentration of your template molecule from plasmid than PCR products.
Another note of caution: PCR products can easily degrade. If your PCR product is made using the same primers as you're going to use for qPCR, then it will degrade to become too short for your primers to work well.
Final note of caution: amplified DNA is a source of a LOT of contamination. Be very careful that you don't contaminate your reactions with your standard curve samples.
Yes you can use a gel extracted PCR band as a standard (make a serial dilution from 10^9 to 10^1) as mentioned before. This method is often used for environmental samples rather than cloning. The reason for this choice, behind the fact it is quicker and cheaper than cloning, it is when you study complex environmental samples (e.g. DNA from soil, water), you have genomic DNA from many microorganisms and a standard from a single bacteria with a specific qPCR efficiency and amplicon size will not match the range of efficiencies and amplicon size/homology find in your environmental sample and will bias you quantification. See the article from Towe et al 2010 that compare both type of standard: Article Differences in amplification efficiency of standard curves i...
To avoid the issue with PCR degradation mentioned by Katie Clark, which will happened within days on such standard, we make the serial dilution of our standard in 1X TE buffer and store the standard in aliquots at -80°C that we use once. This way we don't have any issue with storing our standard.
The gel extraction of your PCR band will remove any primer, buffer (etc), and then ideally you should quantify your product by Qubit rather than Nanaodrop as you will overestimate, but again, it also depend what you want from your qPCR. Working on environmental samples, I focus on relative abundance, i.e. difference between treatments rather than absolute copies number...
As I said this work well for environmental samples, but if you work on DNA from a single organism, then cloning would be the preferred option, although gel extraction would work but my guess is that you are likely to get more criticism when you try to publish your results if working on single organisms.
We used PCR product from gel as a posirive control as a starting point for a validated control. That's what you need!!
Then the follwing steps must be followed
1) cut the band from the gel
2) clean it up with a kit
3) clone the prodcut by AT kloning (kit)
4) selct clones with the insert
5) bulk culture 3-6 positive clones containing the insert
6) isolate the plasmid containing the insert by mini-or maxiprep
7) check the inserts' identy with PCR and sequencing
8) linearise the plasmid
9) characterise the plasmid by preferably qPCR by dilution series
10) determine the correct concentration for use as a positive control (must be in harmony with the expected concentration of the unknown target)
11) aliquot your validated positive control in small and large quantities for use in future
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