DNA Extraction- Crucial Method in the field of Biotechnology research

General statement about DNAextraction:

Juvenile and actively growing fresh leaf tissues were collected for the extraction of genomic DNA. Total genomic DNA was isolated from rice leaves following Phenol: Chloroform: Isoamyl alcohol purification and ethanol precipitation method (Rahman at el., 2007).

Pic: Photo of DNA.Photo Credit: freepik.

Table of Contents

1. What is DNA Extraction?
2. Materials needed.
3. DNA Extraction methods
4. Precaution.

What is DNA Extraction?

DNA extraction, also known as DNA isolation or DNA purification, is a fundamental laboratory technique used to isolate DNA from biological samples for further analysis in various molecular biology applications. DNA extraction is a crucial step in many areas of research, including genetics, genomics, forensics, biotechnology, and other fields where DNA analysis is required.

Materials needed.

Reagents:

Extraction buffer: Extraction buffer is made of combination of Sodium chloride, EDTA, Tris and sodium dodecyl sulfate.

DNA Extraction Methods

DNA extraction is generally done by different methods according to sample.Here I am going to discuss about a general practical laboratory method for DNA Extraction from rice leaf sample.

1. At first, healthy portions of the youngest leaves were cut apart with sterile scissors and forceps
2. Leaves were then washed very well with sterile distilled water and ethanol and dried on fresh tissue paper to remove spore of microorganisms and any other DNA contaminants
3. Approximately 0.2-0.3 inch of leaf tissues were cut into small pieces (as small as possible to facilitate grinding) and taken into a 1.5 ml eppendorf tube
4. 400 µl extraction buffers were added
5. Leaves were grinned by homogenizer
6. Then 400 µl extraction buffers was added
7. Vortexed for 20 second
8. Incubated for 5 minutes at 65˚C
9. Again vortexed for 20 second
10. Incubated for 10 minutes at 65˚C
11. Centrifuged for 10 minutes at 14,000 rpm
12. 600 µl supernatant was transferred in a new tube
13. 600 µl P: C: I (Phenol: Chloroform: Isoamylalcohol = 25:24:1) added into it
14. Again centrifuged for 10 minutes at 14000 rpm
15. Again the upper aqueous layer (about 400 µl) was transferred into a new tube
16. 500 µl P: C: I ( Phenol: Chloroform: Isoamylalcohol = 25:24:1) was added into it
17. Centrifuged for 10 minutes at 14,000 rpm
18. 400 µl upper aqueous layer was transferred into a new tube
19. 40 µl sodium acetate was (1/10th of extract volume) added into it and mixed it gently
20. 1000 µl of absolute alcohol (2.5 times of the total extract volume) was added to it and mixed gently
21. Centrifuged for 25 minutes at 14000 rpm
22. The pellet was observed and the liquid portion of the tube was removed carefully
23. 1000 µl of 70% ethanol was poured into the tube
24. The aqueous portion was removed carefully and the pellet was air dried
25. After removing the liquid completely, the pellets were then air dried and dissolved in 50 µl of TE buffer
26. Finally, the DNA samples were stored at –20°C

Precaution.

During DNA extraction you should aware about bellow mentioned points.

All glassware, centrifuge tubes, glass pipettes, micropipette tips, distilled water and buffer solutions were properly autoclaved to keep away from DNase contamination. Forceps, scissors and tissue homogenizer sticks etc. were sterilized with absolute ethanol.