When we talk about plant genetics and biotechnology, DNA extraction and PCR are rated as the most important and inevitable experiments. Even though, these methods have a long history, they are still enhanced by scientists to perform better and more efficient experiments. This report talks about how you can successfully extract DNA and also perform PCR experiments, especially if you’re a beginner in genetic analysis. These methods help prevent common mistakes that result in the wastage of time and reagents.
RNA extraction kits are used to isolate RNA from DNA. RNA becomes visible in agarose gel as low molecular weight bands. It’s important that you don’t confuse DNA with DNA as RNA influences enzymatic reactions and is degraded by RNase enzyme.
DNA Extraction With Modified Alkaline PVPP Method
This method is good enough to extract DNA from any horticultural plants. The sodium chloride concentration in the extraction buffer is modified in this approach.
Once you’ve prepared all the reagents, you can proceed to weight plant samples. here, you must be careful about the ratio between the weight of plant samples and the volume of extraction buffer. Usually, the buffer ratio is 5 times more than leaf. This ratio can be enhanced to 10 if you’re extracting DNA from harder samples. The highest leaf weight is 1g, if you extract with conventional mortar and pestle, along with liquid nitrogen. The higher the amount of lead, the more difficult grinding becomes. The measurement of grinding is undoubtedly the most significant factors for the successful DNA extraction. Also, it’s important to properly grind the leaves, and give them the shape and form of Japanese Matcha.
DNA Purification By Isopropanol Precipitation
This is one of the most simple methods for DNA purification. Any DNA containing solution can be blended with the same quantity of isopropanol, and then is treated similar to the method ethanol precipitation. High-Salt solution for precipitation is used as the reagent. To carry out this procedure, you must be careful that you don’t touch isopropanol with bare hands. Proceed by adding half volume of DNA solution of the reagent for precipitation and then mix it. Add the same volume of isopropanol as the reagent and mix it. Put the mixture at room temperature for around 10 minutes and centrifuge at maximum speed at 40C. Once all this is done, you can discard the supernatant. Finish the procedure by adding 1ml of 70% ethanol and put it in -800C for around 15 minutes. (Kasajima, Sasaki, Tanaka, Terakawa, & Ohtsubo, 2013)
Tips For PCR Reactions
- Quantity of DNA Samples in The PCR Reaction
Make sure a very balanced amount of template DNA is added in the reaction mixture. 5 ng-is of template DNA is good enough for amplification. Apart from the low-density DNA, concentrations of DNA stocks are usually quite high. DNA stocks are generally diluted by 100 to 1000 times using sterilized distilled water prior to adding it in the reaction. Furthermore, make sure a series of DNA dilutions are compared to find the best dilution scale. However, if it’s cDNA, which is prepared by reverse transcription of RNA, samples must be diluted well in advance of the PCR reaction. This is important due to the components of reverse transcription reaction.
- Using High-Fidelity PCR Polymerase
High-fidely type DNA polymerase are better than the ordinary type, especially when you’re amplifying target DNA sequence from the genome of horticultural plants. PCR has been proved successful in high-fidelity polymerase in rice. Concentration of the target sequence is kept low in big genomes of horticultural plants. This is why higher selectivity of PCR amplification is needed in almost all the horticultural plants.
- Checking The Best Condition Of The Reaction
Apart from the usage of high-fidelity polymerase, PCR is also tested with the gradient annealing temperature from 46 to 680C, with intervals of 20c to 40C. In case the target is not amplified at the given temperatures, primers have to be re-designed. Primer3 software is best for primer design.
The important ingredients to success for DNA extraction and PCR reaction are of great use for all researchers. That said, highly genetic technologies are developed by researchers and all the important experiments like DNA extraction and PCR reaction are inevitable.
Kasajima, Sasaki, Tanaka, Terakawa, & Ohtsubo. (2013). Large-scale extraction of pure DNA from mature leaves of Cyclamen persicum Mill. and other recalcitrant plants with alkaline polyvinylpolypyrrolidone (PVPP). Sci Hortic, 65-72.