 Quality assurance and quality control are critical while working in the molecular biology laboratory. And before proceeding to the downstream applications like PCR amplification, molecular cloning, sequencing and other techniques, we need to measure the concentration of extracted DNA. So in this practical, we will demonstrate that how to measure the concentration of DNA in a sample using nano-drop UV visible spectrophotometer. Basically, nano-drop works on the principle of spectrophotometer which is that absorbance is directly proportional to the concentration and path length. So to quantify the DNA in sample, we need pipettes, pipetips, DNA sample, blank sample which is nucleus-free water. The purpose of nucleus-free water is to blank the instrument. And why we are using the nucleus-free water? Because we have stored the DNA in nucleus-free water. So we will blank the instrument with this nucleus-free water. In the end, we have nano-drop UV visible spectrophotometer. When we will on the instrument, it will take some time to initialize the software. When the program will set up, it will show you different samples which can be analyzed like double-stranded DNA, single-stranded DNA and RNA and even protein sample. So basically, this nano-drop has the LED screen pre-installed in it. And this is the paddle of the nano-drop in which we will pour the sample into the lid. It should be remembered that while performing the experiment, you should clean the tip before pouring the sample. After pouring the sample, you will just close the paddle and run the measurement. Then it will give you the value of concentration of DNA. The system which is installed in this nano-drop is Eclado software which is must sensitive that it can give the concentration of the DNA at the nano-drop level. It also tells us the amount of impurities that are present in our DNA sample. While performing the experiment, first of all, we have to turn on the nano-drop instrument. When the program setup will be initialized, we have to then select the type of nucleic acid molecule for which we are going to measure the concentration. As our sample is double-stranded DNA, so we will select the double-stranded DNA option on the nano-drop. When we will click on the double-stranded DNA, it will show us to load the blank sample. So what we will do, we will open the paddle of nano-drop and clean the both tips carefully. After cleaning the tips, we will load the blank sample which is 1 microlitre nuclease free water. To use it to blank the 1 microlitre of blank sample onto the tip, we will close the paddle of the nano-drop. It will show us to load the sample. We will then load 1 microlitre of DNA sample and then measure the concentration of the DNA. You can see the result. There are 3 ratios. One is nanogram per microlitre which is the concentration of the DNA. Another is the A260 by 280 which gives the purity of the DNA and again A260 by 230 which also tells us about the contamination present in the sample. So in this sample we can see that the concentration of DNA is just 37.4 nanogram per microlitre and the purity of the DNA is about 1.53. Normally the purity of the DNA sample is at 1.8 ratio. This graph also tells us the purity of the DNA. It should be remembered that nucleic acids give maximum absorbance at 260 nanometer. So there will be a peak on near 260 nanometer which will show the presence and then tell us the concentration of DNA. Likewise we can also run the second sample to check the DNA concentration. Again we will clean the tips and add 1 microlitre of DNA sample. Pouring the sample we will close the paddle and click on the measurement. The nano drop instrument will measure the concentration of DNA. And here you can see that in sample 2 the concentration of DNA is 31 nanogram per microlitre and the purity of the DNA is again 1.49 which is A260 by 280 ratio. And these are the other ratios which shows the presence of contamination in the sample. Normally there can be three types of contaminants in the DNA. Phenol, guanidine and protein. If the value of A260 by 280 ratio is 1.8 then it shows that the sample contain the pure DNA. But you can see that here in these results there is contamination of phenol or proteins which just shows that how we can measure or access the contamination through the nano drop spectrophotometer. So that's how we run multiple samples to measure the quality of DNA. Now I will discuss the second case that if there is no DNA present in the sample then what values will be realized on the nano drop instrument. We will again clean the tips with fresh white. Load 1 microlitre of DNA sample. Pouring the sample onto the tip we will carefully close the paddle. In this case you can see the values on the nano drop instrument. We can clearly see the difference between the peaks of these three samples. Like in first sample you can see there is a sharp peak at 260 nanometer. Likewise in the second sample there is again the sharp peak nearly at 260 nanometer. While in the third sample there is no peak present which clearly confirms that in third sample DNA is not present. So we will not proceed with the third sample for our downstream application. So that's how we assure the quality of DNA through nano drop UV visible spectrophotometer. If the DNA concentration is satisfactory then we can proceed for our downstream application like PCR amplification and sequencing. One particular precaution about the nano drop spectrophotometer is that we have to be very careful in cleaning the tips of that instrument because it is much sensitive to detect the concentration of DNA in each sample.