Extraction of genomic DNA from Spinach (Spinacia oleracea) by CTAB method
Aim: To isolate genomic DNA from fresh spinach leaves using the Cetyl Trimethyl Ammonium Bromide (CTAB) method for downstream applications.
Principle: Spinach leaves have a very high content of polysaccharides and polyphenols. These are the real enemies. The cell wall is thick, so we grind it to a fine paste to expose the nucleus. CTAB is a cationic detergent; at high salt concentrations, it stays soluble but keeps the polysaccharides away from our DNA. If the salt drops too low, everything precipitates together and you get a mess. We use chloroform to denature proteins and move them to the organic phase. Finally, isopropanol is used to change the dielectric constant so that DNA becomes insoluble and drops out as a pellet.
Requirements:
Fresh spinach leaves
CTAB Extraction Buffer
CTAB (Cetyl Trimethyl Ammonium Bromide): 20.0 g/L
Sodium Chloride: 81.8 g/L (This is 1.4M, very important!)
Tris-HCl (pH 8.0): 12.1 g/L
EDTA: 7.4 g/L
Chloroform: Isoamyl Alcohol (24:1)
Chilled Isopropanol
70 percent Ethanol
TE Buffer (10 mM Tris, 1 mM EDTA)
Mortar and pestle
Cooling centrifuge
Procedure:
Wash 2 grams of spinach leaves with distilled water and dry them properly.
Grind the leaves in a chilled mortar with 1 ml of hot CTAB buffer until it is a smooth slurry.
Transfer the slurry to a 1.5 ml tube and add more buffer if it is too thick to pipette.
Add 2 percent beta-mercaptoethanol to the tube just before you start. I am knowing this since long: without this, your DNA will turn brown due to oxidation.
Incubate the mixture at 65 degrees for 40 minutes in a water bath.
Add 700 microliters of Chloroform: Isoamyl alcohol and mix by gentle inversion.
Centrifuge at 10000 rpm for 10 minutes.
Take the top clear layer very carefully. To be honest, if you touch the middle white layer, you have to spin it again.
Add 0.6 volumes of chilled isopropanol to the liquid.
Invert the tube slowly. You will see the DNA fibers appearing like a ghost in the tube.
Centrifuge at 12000 rpm for 10 minutes to get the pellet.
Discard the liquid and wash the pellet with 70 percent ethanol.
Let the pellet air dry on the bench until no alcohol smell remains.
Dissolve the DNA in 50 microliters of TE buffer.
Observation: After the isopropanol step, white, cloudy fibers start to aggregate. After the final spin and drying, a small, white or slightly translucent pellet is visible at the very bottom of the tube.
Result: The genomic DNA was successfully isolated from spinach leaves as confirmed by the presence of a high molecular weight band on a 0.8 percent agarose gel.
Conclusion: The method worked because the high salt concentration in our CTAB buffer kept the DNA in solution while the contaminants were removed. Proper grinding is what determines the final concentration you get. If the pellet is not white, it means the washing was not done well enough.
Note: This protocol is for educational use in standard teaching laboratories. Small variations may occur depending on lab conditions and handling. Results may vary. biologynotes.in cannot take responsibility for any errors, misuse, or results obtained. Always follow proper lab safety and instructor guidance.