Protocols for Micropropagation of Woody Trees and Fruits

MICROPROPAGATION OF ELITE NEEM TREE 355
Prepare the master mix as shown in Table 2 and perform the following thermocycler
program for the amplification.
Table 2. Reaction mixture and thermocycler parameters used to amplify genomic DNA in AP-
PCR mode of elite neem tree and its in vitro-raised offsprings.
Reaction components Concentration
Amplification buffer, 10× 1×
MgCl2
2.0 mM
Genomic DNA template 60 ng
dNTP’s mix 200 µM
Primers each 5 pM
Taq DNA polymerase 1 U
Final volume with water 25 µl
Thermocycler program Temp. Time
Initial cycle
Denaturation 94°C 1 min
Annealing 36°C 30 sec
Extension 72°C 1 min
Number of cycle 1
Amplification cycle
Denaturation 94°C 5 sec
Annealing 36°C 15 sec
Extension 72°C 1 min
Number of cycles 35
Final extension 72°C 7 min
2.7.3. Electrophoresis
Analyze the amplification products by electrophoresis in 1.8% agarose gel stained
–1
with 0.5 µg ml ethidium bromide.
Agarose Gel Preparation. Prepare agarose gel by melting 4.5 g agarose powder in
–1
250 ml 1× TAE buffer, cool it to 60°C and before casting add 0.5 µg ml
ethidium
bromide to the gel. Load the gel with PCR products and use 10 kb DNA ladder as
molecular standard. Run the electrophoresis, visualize the bands under a gel documentation
system and take the photograph of the DNA bands. As expected, a
uniform pattern of monomorphic bands is seen with primer OPA-13 (Figure 4c) and
primer OPC-14 (Figure 4d) indicating no variation among the mother plant and 13
micropropagated offsprings.