ADVERTISEMENTS:
In this article we will discuss about the DNA extraction and amplification.
Normally, CTAB (cetyl trimethyl ammonium bromide) method is used.
However, SDS method is also common and is described as follows:
ADVERTISEMENTS:
1. The leaves are washed with sterilised distilled water and blot dried and cut into small pieces with a sterilised blade.
2. 1g of leaf material is placed into a pre-cooled mortar.
3. Liquid nitrogen is added to it and a fine powder is made and transferred to Oakridge tube/eppendorf tube (eppendorf tubes are appropriate for 1g leaf powder).
4. After making a fine powder 800 µ extraction buffer sodium dodecyl sulphate (SDS) is added.
ADVERTISEMENTS:
5. The sample is homogenized and incubated in water bath at 65°C for 1 hr.
6. 300 µ1 potassium acetate and equal volume of chloroform: isoamyl-alcohol (24: 1) are added.
7. The sample is centrifuged at 4°C and 12,000 rpm for 30 min.
ADVERTISEMENTS:
8. The supernatant (200-300 µ1) is taken into another eppendorf tube of 1.5 ml vol. and equal volume of isopropanol is added.
9. The sample is centrifuged at 4°C and 12,000 rpm for 20 min. and the supernatant is discarded.
10. The pellet is washed with 70% ethanol (approximately 200 µ1).
11. After washing the eppendorf tube is kept in inverted position over paper towel/tissue paper for removal of ethanol leading to drying of DNA.
12. The pellet is dissolved in 250 µ1 TE (tris EDTA) buffer and sample is kept at – 20°C overnight/till needed for PCR.
ADVERTISEMENTS:
13. After storing overnight at – 20°C 4 µ1 RNase and 4 µ1 proteinase K are added to each sample.
14. The sample is shaken gently through tapping by finger and incubated in water-bath at 65°C for 1hr.
15. Equal volume (approximately 250 µ1) of chloroform: isomylalcohol (24: 1) is added and the sample is centrifuged at 4°C and 12,000 rpm for 35 min. and supernatant is transferred by micropipette to another eppendorf tube.
16. Step 15 is repeated on freshly collected supernatant 2-3 times in order to remove phenolic compounds.
ADVERTISEMENTS:
17. The supernatant is taken and 3 M sodium acetate and ethanol are added.
18. The sample is centrifuged at 4°C and 12,000 rpm for 35 min.
19. Pellet is dried with paper towel and dissolved in TE (Tris EDTA) buffer. The sample is now ready for PCR.
20. The conditions vary according to crop and primer and the system standardised. Normal system usually giving satisfactory results across a range of crops at Pantnagar is as: Denaturation: 94° C (5 min), annealing: 35° C (1 min), extension: 72°C (5 min) and number of cycles: 40.
ADVERTISEMENTS:
21. The amount of template and primers used for 25 µ1 of PCR reaction mixture are 20 ng DNA (2µ1) and µ1 of primer.
22. The concentration of Taq DNA polymerase used in PCR is 0.5 units/25 µ1.
