“DNA extraction is a process of isolating or getting DNA from a cell or population of cells using chemical or physical methods.”
~1.80 numbers we want every time we extract DNA! Why is it important?
DNA absorbs UV light at 260nm. By taking the ratio of 260/280 we can check the purity of DNA. A pure DNA has a 260/280 ratio of nearly 1.80 and that is why this number is so important.
However, to get pure DNA using conventional and manual DNA extraction protocol is quite a tedious job. A lot of optimization and experience is needed to play with chemicals and the protocol.
In the present article, we will give you some of the tips that are very secret and proven by us, to get a good DNA during extraction. But before that let’s understand some basics of DNA extraction.
Key Topics:
What is DNA extraction?
Isolating DNA from the rest of the cell organelles is known as DNA extraction.
DNA with a quality of 1.80 and a quantity of nearly 150ng is required in every genetic technique starting from PCR to restriction digestion and from DNA sequencing to DNA microarray. So it is clearly indicating that we need to learn DNA extraction in a proper way if we want to go ahead in genetics.
DNA is located in the nucleus of a cell. To isolate it we have to first break the cell wall and then the nuclear envelope. In the last step, we have to remove all other debris left.
So to do this various combinations of chemicals and solutions are prepared. For instance, a saturated phenol can digest protein portions of a cell. Alcohol precipitates the DNA and separates it.
However, for making a DNA extraction protocol effective we need to do optimizations. But the question strikes in your mind is that, if ready to use DNA extraction kits are available, then why do we need to do all this stuff?
Firstly, as a student or beginner, you should learn the manual DNA extraction process. You can understand how different chemicals work and what their role is. Until and unless you don’t do it, how you can understand the composition of chemicals used in different DNA extraction kits.
Second, the manual DNA extraction methods are cheaper than the ready to use kit. I mean it is way cheaper.
In my opinion, the yield of manual DNA extraction kits is a lot more. For DNA microarray like experiments, only high yield DNA is required. So for doing DNA extraction for DNA microarray you have to do manual DNA extraction.
Some common DNA extraction methods are enlisted here:
- Phenol-chloroform DNA extraction method
- Proteinase K based DNA extraction method
- CTAB DNA extraction method
These three are the most common methods used so often for DNA extraction. If you want to learn more about the principle, protocols, and how different chemicals are used in it, we have covered separate articles on it.
You can read it here: Different types of DNA extraction methods.
Now come to the important topic of this article. Tips for DNA extraction. Let me first tell you that these tips are used by me and my team members in our lab. So it is 100% practically working. You can use it directly.
10 Secret tips for DNA extraction
Choosing the right DNA extraction method
Not all the methods are used for all types of cells. For example, the plant cells have harder cell walls rich with pectin, in order to isolate DNA from it, phenol-chloroform DNA extraction method alone can’t work. For that, we have to use the CTAB method with or without proteinase K.
So first, you have to decide which DNA extraction method goes well for your specimen. Some of the cells, sample types and extraction method for them are enlisted here:
- Phenol chloroform DNA extraction: blood and solid tissues
- Proteinase K method: Blood, amniotic fluid, chorionic villi, saliva and buccal smear.
- CTAB: plant sample
- Triton X 100: bacterial and plasmid DNA extraction
- Nonidet P 40: DNA extraction from blood.
Note: In some cases, you can combine two different methods for gettin’ good results. For example, in plant DNA extraction using both proteinase K and CTAB methods gives excellent results. However, the accuracy of the results depends on the expertise and experience of the researcher.
The right combination of chemicals
Combination, quantity, and weighing of chemicals are very very important in genomic research. See we are dealing with very small amounts of chemicals so we need to be a little sharp in weighing chemicals.
Trust me if you weigh each chemical and properly, as per the protocol, set pH, and prepare the solution correctly, your experiment will never be failed. That is my personal guarantee.
However, in order to optimize the method, try to use different combinations and select with suites you.
Gettin’ precipitate
The DNA is visible only in the form of a precipitate. If precipitates are not observed, our experiment fails. However, that doesn’t mean there is no DNA in the tube.
Always try to obtain precipitation and the right type of alcohol is key for that. For instance, to precipitate DNA we can use any type of alcohol, though, less precipitation is obtained by using methanol instead of isopropanol
Pro-tips: Use the 70% isopropanol and 1/10 volume of sodium acetate to precipitate DNA.
Chilled alcohol
Yet less known but proven tips is the use of chilled alcohol. Precipitate the DNA using pre-chilled alcohol. Notwithstanding, there is less scientific evidence and explanations are available to explain why we are using the chilled alcohol.
But trust me it works. You can try it. Precipitate one tube with normal alcohol and another with the chilled alcohol. You will see the difference.
Washing DNA
After getting a precipitate we have to wash our DNA to remove the traces of other chemicals and debris. Two washing steps are generally recommended. But let me tell you that to get pure DNA, wash the DNA three or four times with alcohol.
Use 100% alcohol for washing. Perform centrifugation with 10,000 rpm.
Pro-tips: wash the DNA until you get a white-clear DNA pellet.
Use less and get more!
We always think that using a large amount of sample is a key to get high DNA yield but it is not true. Use less amount of starting sample approximately 0.5ml to 1ml and process it properly, trust me you will get good DNA yield.
Mixing the sample
Small things make big differences in genetics. How you use your chemicals, mixing, inverting, resting and other things have a great impact on final results.
Mixing is also as important as choosing the protocol. When we mix a sample with chemicals or solutions, the chemicals pass through the DNA and remove debris, digest proteins, and remove other cell organelles.
When you add a chemical to the sample, mix it properly and gently so that every chemical can work finely as well as our DNA remains intact.
Note: never vortex the DNA sample, it makes DNA fragmented.
Dissolving the DNA
Use the TE buffer to dissolve the DNA always. Our TE buffer recipe is given into the article: Importance of TE buffer in DNA extraction. The combination given in it is our own, proven, and best to use.
A precipitated DNA can’t be directly used for downstream processing. We need to dissolve it first. Water can be used for that, though, TE buffer is the best choice to do it.
Notably, in some assays, the EDTA present in a TE buffer hurdles the reaction. To avoid it, use the nuclease-free water to dissolve the DNA.
Store it properly
Extracting DNA is a time-consuming process. It takes around 2 to 3 hours to extract DNA. So it is obvious we can’t perform PCR or sequencing on the same day. Also doing PCR, sequencing or microarray is also costlier therefore we have to store our DNA sample.
DNA can remain stable for thousands of years but to preserve it in the intact form we have to follow some instructions.
To store a DNA, always dissolve it in a TE buffer. Store at -20°C for short term and -194°C for long term storage in liquid nitrogen. Make different aliquots of DNA.
Conclusion:
To learn more about DNA extraction you can read our previous article which has all the information on it. Read it here: Different types of DNA extraction methods.
Extracting DNA is not as hard as you think. But to master skills, you should learn every time something from your DNA extraction protocol. I have performed DNA extraction thousands of times and still, I learn something every time when I do it.
All the technical information like the concentration of every chemical, how to make DNA extraction buffers and chemicals used in it are explained in our previous articles, that is why we have not given that information here. Conclusively, we can say weigh chemicals properly and mix the sample gently, these two things are key to success in DNA extraction, in my personal opinion.
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everything is explained beautifully and in detail.. thank you so much