The qPCR protocol was as follows
|Ssofast Evagreen MM||10||120|
|Nuclease Free H2O||9.8||117.6|
1. Added the reagents from highest volume to lowest to the master mix
3. pipetted into .5 ml qPCR tubes
4. Added 0.2 ul RNA to each tube from each isolation
5. Placed into the Opticon 2 qPCR machine
6. Ran the program "Sybr New Plate + Sybr cDNA 55 melt 2 reads"
As you can see, the samples began amplifying at 27-30 cycles which is indicative of a genomic DNA contamination. The negative control did not begin amplifying until 37 cycles.
For future work, I need to use a DNase treatment to remove genomic DNA before I create cDNA. Next week I will isolate the 310 heat shock animals, DNase treat everything, and produce cDNA.