P-FPMI-276 - P-FPMI-276
RNA isolation protocol:
5 biological repeats of Pseudomonas aeruginosa and 5 biological repeats of Pseudomonas aeruginosa parR gene knockout were grown in BM2-glucose media containing 2mm MgSO4 and 4 ug/ml indolicidin. For each 12.5mls of culture grown to an OD600 of 0.5 was harvested by centrifugation and the pellets were used for RNA extraction. 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, noexpections 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