DNA extraction Method for plant sample using CTAB method

1. | DNA Extraction Method

2. CTAB Method
Abbreviated: Cetyl trimethylammonium bromide
Function: CTAB buffer is used to lyse plant cells in the same way SDS is used to lyse bacterial
cells. They work in a similar fashion, apart from CTAB being a cationic detergent while SDS is
anionic.
Procedure
1. Warm the CTAB buffer at 65oC for 15 min (for its activation)
2. Plant sample is taken in 200mg and stored at -80oC (to freeze the sample, makes it ready
to be broken easily)
3. Place the frozen sample and CTAB in mortar pestle and grind it (During the process add
200ul of CTAB gradually up to 600ul)
4. Collect the resulted sample from mortar and put it in Eppendorf tube (Place all of it)
5. Put the Eppendorf tube in water bath at 65oC for 45 min
6. Then centrifuge the Eppendorf tube at 12000rcf/g for 15 min (Two layers will be formed)
7. Pick the supernatant and put it in another Eppendorf tube
8. Add chloroform and isoamylalcohol in 24:1 (Make in equal amount to supernatant |
these chemicals are used for nucleic acid precipitation)
9. Repeat step 6
10. Now the supernatant is clear. Pick the supernatant and place it in another Eppendorf tube.
11. Add isopropanol in equal amount to supernatant (For precipitation or separation of
DNA from RNA)
12. Store the Eppendorf tube overnight at -20oC
13. Repeat step 6
14. Now pellet will be formed containing DNA
15. Discard isopropanol from Eppendorf tube carefully
16. Add ethanol to precipitate DNA. Afterward, discard the ethanol and let the pellet be dried
for an hour
17. Now add TE buffer in 50ul for pellet dissolution (this process can be done by heating or
pipetting or tabbing or inversion)
18. To check the integrity (stability) of DNA, gel electrophoresis is done