I explained the principle behind the extraction of pDNA in my last post. Extracted the DNA, what next?
Let us quantify the DNA extracted.
How to quantify? Very simple, all you need is Spectrophotometer.
- Just take 1 microliter of your DNA extract in an eppendorf, make it upto 500 microliter with nanopure water. (else 1000, or any desired volume)
- mix it well
- Now, put this into a quartz cuvette (0.5ml), measure the O.D value at 260 nm. Use blank as nano pure water.
- Note: The path length of the cuvette must be 1cm.
It's very simple, do the following calculation.
If your O.D. Value is 1, then, the concentration of your DNA sample is 50 microgram/ml
so, now, if your concentration = obtained O.D.( let it be 0.5) * 50 microgram/ml * Dilution factor
How to calculate the dilution factor?
Simple! here your dilution factor is 500/1 i.e., 500 micro liter contains 1 micro liter of DNA, you can calculate according to your dilution.
Now, using this calculation you can get the concentration of your DNA sample! Very simple, isn't it?
DNA has absorption maximum at 260 nm; that's why we are taking the O.D value at 260 nm. This absorption maxima at 260 is due to the presence of nitrogenous bases in DNA.
Proteins usually have the absorption maxima at 280 nm.
DNA is hyperchromic - i.e., the O.D value increases with denaturation of DNA. i.e, single stranded DNA absorbs more than double stranded DNA.
How to check the quality of your DNA sample?
Take the O.D value at 260 nm as well as 280nm .
Calculate O.D 260 / OD 280.
If it is equal to 2, then your sample is 100% pure!
If you get values below 2, there is contamination with protein.
Understood? Any doubts? Feel free to question me!
Thanks, Bye, Wish you a happy time!