Determining DNA Concentration (BIOL310)

In this video, I walk you through the process of determining the DNA concentration using absorbance measurements at 260nm. We then combine those with a reading at 280nm to get idea of the level of purity of the DNA in our sample.
0:00 - Introduction
0:32 - Preparing dilutions of our samples
2:13 - Selecting an appropriate blank
2:33 - Taking absorbance readings
3:23 - Calculating the concentration and level of purification

[@mohamadjaddo3968]

Exactly what we needed, not everyone has a Nano drop so this video was very much appreciated. well done.
الله بجزيك بالخير

[@ItsLearnable]

Thanks

[@lolo6172]

Thank you very much that was easy and very helpful.

[@ItsLearnable]

Great to hear!

[@lona6061]

Excellent explanation

[@ItsLearnable]

Thanks

[@lona6061]

In the calculation of concentration of the sample, what does 50ul denotes? Vol of what? Thankyou

[@ItsLearnable]

Thanks for asking the question @@lona6061. It's nice to see people trying to figure out *why* things are done in a particular way. 👍
If you're referring to this: czcams.com/video/O8FytCl_99Y/video.html
then please note that it's not a volume. The "50" here is a constant - it's actually the inverse of the extinction coefficient of DNA [0.020 (µg/ml)-1 cm-1 ], and we're using the Beer-Lambert equation to determine the concentration. According to the equation: Concentration = Absorbance / (extinction coefficient * path length). And we have to multiply that by the dilution factor to get the actual original sample concentration.
For many students, in what is an introductory course to molecular biology methods, the above might be a bit scary/intimidating. That is why we generally just simplify it, as I showed in the video.

[@lona6061]

Now it's super clear! Learning and preparing for research watching your videos

[@mnly.h9606]

Thanks for your wonderful video
How much solution do you take of each diluted microtube (100× / 1000×) in order to measure the absorbance in spectrophotometer?

[@ItsLearnable]

We use the full amount (1ml) because of the type of cuvettes we have.
There are some cuvettes that have a 0.1cm pathlength, which allow you to use much smaller volumes to measure your DNA or RNA samples, but we don't have those cuvettes in our teaching lab.

[@ItsLearnable]

using a cuvette with a smaller path-length would also require you to adjust your calculations to account for that. The calculation we used, assumes a path length of 1cm (this is standard for most cuvettes)

[@mnly.h9606]

@@ItsLearnable Thank you so much 🌹🌹🌹🙏🙏🙏

[@omaral-jarallah4808]

Can you use fluorometer instead of spectrophotometer to measure the DNA concentration?

[@ItsLearnable]

DNA, on its own, does not fluoresce.
While it is true that we use UV light to make it "glow" on a gel, that glowing is not because of the DNA itself, but because of a common additive in agarose gels called ethidium bromide, which binds to DNA while it migrates in the gel.