Cfx96 real time system c1000 thermal cycler machine

RNA was isolated from Daphnia after heat shock after indicated recovery times (as well as a control, non-heat shocked sample). cDNA was synethsized as described before using random hexamers. We first standardized our cDNA by performing real time PCRs with serial dilutions of cDNA to ensure appropriate efficiency and correlation. Every reaction was performed in triplicate in a total volume of 20 μl. This included 4 μl cDNA, 250nM Hsp70 or β-actin primers, and SsoFast EvaGreen Supermix (BioRad). β-actin was used for normalization. All reactions were run on a BioRad CFX96 Real Time System C1000 Thermal cycler machine with the following conditions: 95° C for 30 seconds, 95° C for 5 seconds, 52° C for 5 seconds (the last three steps repeated for 40 cycles), 65° C for 5 seconds, and then 95° C for 5 seconds. We analyzed our data using the Bio-Rad CFX Manager Software and used the 2^−ΔΔCt method to compare Hsp70 expression in heat shocked versus the non-heat shocked samples. Note that 3 separate RNA isolations were utilized from 3 separate groups of Daphnia to serve as biological replicates for each clone for each treatment.