By: N. Shreya Mohan (MSIWM042)
AIM– To isolate the DNA from a plant sample
THEORY– Plant extractions for DNA is considered one of the tedious methods for high quality DNA isolations. Unlike animal tissues, which have the same tissue type in different species, plant tissues structural biomolecules and metabolites keep changing. Polysaccharides and polyphenols are two very special class of biomolecules that are very different from species to species and thus, becomes a hurdle during DNA isolation. Those biomolecules which contaminated drastically affect the manipulation of DNA isolation.
- 2gm of plant sample was taken in a motor and 5ml homogenization buffer was added. Then, it was ground well for 15 minutes. Then wash it thoroughly.
- 15 ml of lysis buffer was added, and it was again ground well for 15 minutes.
- The mixture was incubated at 65℃ for 30 minutes in microcentrifuge tubes.
- The tubes were then centrifuged at 8000 rpm (rotations per minute) for 10 minutes at room temperature.
- 500µms supernatant was taken in microcentrifuge tube and equal volume of PCL mixture was added.
- Again, tube was centrifuge at 12000 rpm for 10 minutes.
- Then, the upper aqueous layer was collected (50 µL) in a new tube and chilled ethanol 200 microliter was added.
- Tubes were incubated at -80℃ for 10 minutes or at 4℃ for 1hour.
- Tubes were centrifuged at 12000 rpm for 12 minutes.
- The supernatant was removed, pellet was air-dried and this is dissolved in µL of TE buffer.