I'm planning to extract genomic DNA from whole blood using a commercially available kit (Preferably QIamp) and then do a PCR reaction to amplify the gene of interest. I am not a molecular biology person, so it has been a baffling question for me to realise how to find the concentration of the extracted DNA as we then have to add a specific weight (amount-few micro/nano grams) of DNA to the PCR reaction which act as the initial template. Can anyone offer me an inclusive approach to determine the concentration of DNA? Please note I will probably have to deal with quite a number of samples within a very short time, so it has to be a convenient procedure.
I think you can do gel electrophoresis and measure the nanodrop using spectrophotometer.
Electrophoresis in 0.8-1.2% agarose gel will show quality, weight, and amount of DNA (and RNA) in you samples.
No, it is not neccesssary to run your samples on gel (not to say before the measurement), however it may help you to determine the quality of your DNA.
Usually one determines the DNA concentration using spectrophotometry, either in conventional setup or with what is called nano-drop. Advantage of nano-drop is that you need only 1-2 ul of your sample and and it's quite faster as you do not have to clean the cuevette each time. However, it is less accurate (but still good for PCR, unless your concentration is too low).
However, what we usually do is to run the PCR with primers to housekeeping gene (in our case actin) and by the results adjust the amount of DNA added.
electrophoresis followed by nanodrop is enough to know quality of DNA extracted...
Dear friend, as you mentioned that you are not expert in molecular study I would like to suggest before starting your study, you had better study more about pcr, why we need to know about DNA concentration, what is contamination in pcr and it's effect on our work. What is m1v1=m2v2 and it's application. Also, I would like to suggest you have a look on www.protocol-online.org in molecular biology part. As you can see above, our friends have discussed valuable issues but you had better start studying on the basic matters in molecular biology then read their advise. As you mentioned you are not familiar with mol biology.
Good luck
I think the best method is Nanodrop Spectrophotometer technique
you can get the DNA concentration and purity, I have tried it before
Best regards
Appreciate your more comprehensive and detailed contribution, Dr Wathsala. I am not quite sure whether we do have a nanodrop at our place. But If it exists, I will prefer to use that instead of running all the extracted DNA on a gel. If not, I'd stick with a conventional spectrophotometer method which may not be as easy as the nanodrop, but at the end of the day, they both will give me the absorbances. So I can do the downstream calculations to find out the concentration. Thanks again.
The previously mentioned Nanodrop instrument is a good start, as it is easy to use and gives both a concentration and an idea of quality. Unfortunately, it's based on spectrometry and therefore is not very sensitive (values below 20 ng/ul are unreliable in my hands) and is sensitive to contaminiation of organic compounds (including small amounts of ethanol and isopropanol, commonly used to precipitate DNA after isolation). An alternative can be Life Technologies' Qubit (http://www.lifetechnologies.com/ch/en/home/brands/product-brand/qubit.html). This machine relies on dye incorporation, and is therefore very sensitive (I can reliable measure concentrations of less than 1 ng/ul) and is not sensitive to contamination (it can even discriminate between double stranded DNA, single stranded DNA and RNA). Unfortunately, it comes with some disadvantages as well: it doesn't give an indication of the quality and the dye that needs to be added to each sample costs in the range of $0.50 per sample. In my experience though, the benefits of the Qubit greatly outnumber the disadvantages though.
If you like to use the Qiagen kit with whole anticoagulated blood simply follow the instructions from the manual and read the table of the average amount of DNA guaranteed per extraction. This is your mark. Endpoint PCR should work in each case with 1 to 5 µl of the eluat. If your downstream application is quantitative wether electrophoresis and gel staining nor nanodrop is sensitive enough. And I wouldn't recommend an unexperienced person to start with quantitative applications.
Hey harindra, Nano drop is the best method to know the concentration of DNA extracted, as its really easy to use nanodrop and no. of samples can be quantified in very short time, and harindra i would like to mention its not weight its concentration (template DNA) you are going to use in PCR. Good luck
Dear Harindra
Plz accept this book as a gift from me. it will help you a lot for PCR and downstream procedures.
Best Regards
there are many methods,
1) Agarose gel electrophoresis. If you have a control sample with know concentration, you can quantify in a gel documentation system (we have a biorad geldoc)
2) Spectrophotometer.:- You can prepare 1:50 dilution of the samples and then quantify it in a UV spectrophotometer and calculate the concentration of the DNA from the OD reading(OD of 1 corresponds to 50ng of DNA)
3) Nanodrop / Nanoview: very little DNA required for quantification
4) Qubit: most sensitive in quantifying DNA but requires optimisation every time
If you are just going to do normal PCR, spectrophotometer reading or even a gelcheck is enough. But if you are going to use the DNA for real time PCR, quantification is critical and you might need to go for nanodrop / nanoview or qubit.
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