This was done to really test the detection methods we’re using for gDNA contamination in our qPCRs. qPCR plate layout/set up is here. Anneal temp 50C.
It should be noted that the 07:12-08 RNA was diluted 1:10 before adding to the rxns to make it similar concentration to the DNased RNA. Also, ran out of the gDNA when adding to the 16s primer rxn. Used 1uL of H2O to “wash” the stock tube and added that in hopes of still detecting something. Unfortunately this was the only stock of H.crach gDNA that came up positive in yesterday’s rxns. “Dirty” cDNA is cDNA made with DNased RNA determined to still have gDNA using 16s primers AFTER the cDNA was already made. “Clean” cDNA used DNased RNA determined to be free of gDNA BEFORE the cDNA was made.
Signals were present with 16s primers in the following samples:
RNA 08:3-7, clean and dirty cDNAs and gDNA.
The other two primer sets only showed signals in the RNA 08:3-7.
Nothing came up in any of the DNased RNA samples.