Dna Isolation Lab Report

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3.1 DNA Isolation: Genomic DNA of Bacopa Monnieri was isolated using conventional CTAB method of Doyle and Doyle (1990) with minor modifications. About 2gm of young or recently matured leaves were collected and washed with distilled water and were then blotted dry. It was then ground into a fine powder in mortar and pestle using liquid nitrogen and PVP. Due care was taken to prevent the thawing of the material. The powdered material was immediately transferred to an autoclaved eppendorf tube with 800μl of extraction buffer (2% CTAB) with 0.2% β-mercaptoethanol (i.e.20μl). These sample tubes were then incubated at 65˚C in water bath for one hour with gentle end to end inverting of each tube at an interval of 10 minutes. The tubes were cooled to room temperature and added equal volume (800μl) of Chloroform: Isoamyl Alcohol…show more content…
ISSR primers were used to amplify the DNA using thermal cycler. The standardized PCR reaction conditions were: initial denaturation step at 94°C for 5 minutes followed by 36 cycles comprising of denaturation step at 94°C for 1 minute, a primer annealing step of 55°C for 1 minute and an extension step at 72°C for 2 minute terminated with a final extension at 72°C for 7 minutes and final hold at 4°C for infinity. S.no. Reagen Reagents Volume 1 (10X) Dream Taq assay buffer 2.5 μl 2 25mM MgCl2 1.5 μl 3 (10X) dNTP Mix 0.5 μl 4 ISSR primer 3 μl 5 Taq polymerase 0.1 μl 6 Template DNA 2 μl 7 Distilled water 15.4 μl Total 25 μl Table: PCR ingredients for 25 μl reaction S.no. Marker Sequence 1 ISSR 1
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