Diseases and Management of Crops under Protected Cultivation

(Diseases and Management of Crops under Protected Cultivation)
as a template. CTAB method is used for isolation of genomic DNA from plant pathogens. Also
liquid Nitrogen is used for grinding the mycelial mats, for effective disruption of the cell wall and
cell membrane. Template DNA should be pure i.e not having any contamination of RNA and
protein.
Ribonuclease enzyme is used (as per the protocol) for removal of RNA contamination from
the genomic DNA of target plant pathogen. Quantification of the purified genomic DNA was done
by checking the UV absorbance at 260 nm with the help of U.V. spectrophotometer. O.D. was also
taken at 280 nm to calculate the ratio OD260/OD280. The ratio gives the amount of RNA (or)
protein in the preparation. A value of 1.8 is optimum for best DNA preparation.
Taq DNA Polymerase- Thermus aquaticus DNA polymerase (Taq DNA polymerase) is a
thermostable enzyme that replicated DNA at 72-74 ° C and remains functional even after incubation
at 95 0 C. The enzyme has 5’-3’ polymerase activity and 3’-5’ exonuclease activity.
DNTP’s- The deoxynucleotidetriphosphates are dATP, dGTP, dCTP, dTTP (used as 10 mM
each).
Assay Buffer (10X)- 10 X assay buffer for Taq polymerase enzyme. Assay buffer contains 10
mM Tris-HCl (pH 9.0), 15 mM MgCl2, 50 mM KCl and 0.01% gelatin.
Standardization of the PCR Conditions
There are number of variables in a PCR which have to be optimized to give target
amplification. These parameters are:
• Denaturation temperature and denaturation time
• Annealing temperature and annealing time.
• Amounts of template and primer to be taken
• Concentration of MgCl 2 in the assay buffer
• The number of Cycles to be proceeded.
Preparation of PCR mixture: A master mix containing all the above mentioned components and
no template DNA should be prepared in laminar airflow under sterile conditions for total no. of PCR
tubes to be used. This will reduce the pipetting errors. Then distribute the master mix in each tube
(24 µl each) and finally add 1 µl of different DNA template in each tube. Gently mix and centrifuge
the mixture for 10 sec. Annealing temperature should be standardized using T-gradient
programme.
Analysis of the Amplification (PCR) Products: Analysis of the amplicons will be done by
Agarose Gel Electrophoresis in a submerged gel electrophoresis unit (for fractionating RAPD
markers on agarose gel). Prepare Agarose gel (2.0%) by dissolving appropriate amount of
agarose in 0.5X TBE buffer. For each well, mix DNA loading dye and DNA samples in 1:6 ratio
and load with a micropipette. Start Electrophoresis at 80V for 3.5 hrs in 1.0X TAE electrophoresis
buffer. Stain the gel in ethidium bromide solution. After destaining in deionized water, view the gel
image in U.V. transilluminator and store in gel documentation system.
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