The for 45 sec, SSR primer was

The PCR tubes containing sample were placed in PCRthermo cycler machine and all the requirements about temperature stages and no. Of cycles were fed in the software of machine and the reaction was started first preheating the lid to increase the temperature of upper portion to avoid evaporation of the sample from PCR tubes to the upper portion. After denaturation at 94 ºC for 45 sec (Rajapakse et al., 1992 ). Primer annealing for 45 sec, SSR primer was used with 58 0c annealing temperature and finally extension for 1 min with completion of 1st cycle and similarly all steps were repeated for 35 cycles with final extension for 7 min and at end storing PCR samples at 4 ºCtemperatureThe two above stated quantities were mixed together in a flask. The flask was placed in microwave oven for 2 min. The flask was removed from the oven and 8 µl of ethidium bromide was added into the solution after making it some cold.care was taken because ethidium bromide is carcinogenic. The gel was smeared into the gel assembly tray with comb adjusted in it. The bubbles were removed with the help of piece of paper. It was left for solidification (Boddey et al., 2000).1.      2 to 3 leaves of sample (Sugarcane leaves) were taken in mortar and with the help of pestle they were ground into fine powder in the presence of liquid nitrogen.2.      This fine powder was filled into the falcon tube and 15 ml of pre heat 2x CTAB buffer was added into the falcon tube and it was shaken gently for mixing the contents3.      These falcon tubes were incubated into the water bath at 65 ºC temperature for 30 min. During the incubation period the contents of the falcon tubes were mixed three times by removing from water bath and shaken well by hand.4.      After incubation period 15 ml of chloroform: iso-amyl alcohol (24: 1, v: v) was added into the sample and it was placed into the centrifuge machine at 20 ºC temperature and 4000 rpm for 30 min (Yamada et al., 1998).5.      The centrifugation resulted two separate layers of the contents in falcon tube, an aqueous layer (supernatant) and a layer of debris in the bottom of falcon tube. The supernatant was transferred to another falcon tube with the help of micro pipette.6.      Equal volume of chilled 2-propanol was added into the falcon tube to precipitate DNA. The sample was placed in centrifuge machine at 4 ºC temperature and 4000 rpm for 15 min. This resulted DNA to precipitate in the form of pellet in the base of the tube separated from aqueous layer.7.      Aqueous supernatant layer was discarded and the pellet was washed with 70% chilled ethanol. The pellet was dried on tissue paper in laminar flow for about 3 hours and it was dissolved in 500 µl of de-ionized water in falcon tube8.      The suspension was transferred into a new 1.5 ml micro-centrifuge tube (eppendorf tube) with the help of micro pipette and 7 µl of unease (10mg/ml) was added into the tube to digest RNA. The sample was incubated at 37 ºC temperature for one hour in the dry bath.9.      After incubation period an equal volume of chloroform: iso-amyl alcohol (24: 1, v: v) was added into the sample and it was placed into the micro-centrifuge machine at 20 ºCtemperature and 13000 rpm for 15 min for phase separation. This resulted into an aqueous supernatant separated from debris in the base of eppendorf tube.10.  The supernatant was transferred to another 1.5 ml micro-centrifuge tube with the help of micro pipette and 30 µl (0.1 volumes) of 3m NACL was mixed into the sample by gently shaking the eppendorf tube.