P-FPMI-266 - P-FPMI-266
Dendritic swarm colony growth (2 to 3 mm of the swarm edge) was harvested by a sterile inoculating loop and resuspended in BM2 swarming media supplemented with RNA protect reagen (Qiagen, Germany).
RNA was extracted using the Qiagen RNeasy midi kit cat #75144, the isolation was carried out using the protocol "Isolation of Total RNA from Bacteria" in the handbook supplied with the kit. No changes were made in the protocol.
RNA was treated with DNA free kit from Ambion (cat #1906). Samples were incubated at 37C for 30 minutes, no expections were made to the protocol
To ensure removal of gDNA, RNA was checked on as agarose gel and control pcr using rpsL primers was done using genomic PAO1 DNA as a positive control, RNA samples showed no PCR product.
Amplification, Extracted product