Cdna Synthesis Lab Report

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The first strand cDNA was synthesized using 5µg of total RNA. The RNA and primer mix was incubated at 65°C for 15 min and then the tube was placed on ice for 5 min for chilling. Subsequently, 200 ng of gene specific primer, 5µl of RT-Buffer (10x), 0.25µl of RNasin (40U/ µl), 2 µl of dNTP mix (2.5 mM each), 5µl of DTT (0.1M), 2 µl of MMuLV RT (20U/µl) was added and the final volume 50 µl of reaction mixture was prepared using DEPC water. Reaction mixture was incubated at 42°C for 1 h and the reaction was terminated by heating at 94°C for 5 min followed by snap-chill on ice. The first strand was used for the PCR with gene specific primer for the second strand synthesis followed by cDNA. Further, reverse transcriptase - PCR (RT-PCR) was performed…show more content…
All reactions were performed in triplicates on Applied Biosystem Step-one Real Time PCR machine and the resultant data was subjected for melting curve analysis to check for the presence of nonspecific products. This experiment was performed in triplicates and the quantity of the hsp90 and hsp70 mRNA was normalized against transcript quantity of tubulin gene in non-heat shocked larvae of B. mori (Pfaffl, 2004).
2.6. Analysis of biological and commercial traits
Heat shock response of male and female larvae during different instars subjected for HS at different temperatures was assessed based on percent of total mortality (inability to enter succeeding instars or to spin cocoon). In order to evaluate the fitness of silkworm larvae exposed to different HS temperature based on number of cocoons harvested from the number of larvae brushed (ERR-effective rate of rearing). Further, physiological output was measured based on cocoon weight, shell weight and shell ratio which are derived following standard formula. All these data were statistically analyzed using the ANOVA
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