Total RNA isolation from Bacterial Cells
To isolate total RNA from the given bacterial culture
Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol (Sigma). Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol. RNase enzymes can be inactivated by including diethyl pyrocarbonate (DEPC)
• Bacterial culture
• Isopropanol solution
• TAE buffer
• 70% ethanol
• Take 800 μL of bacterial culture in a fresh Eppendorf. To this add 160 μL of Trizol (1/5th of culture volume).
• The solution was mixed well by pipetting several times. To this add 32 μl of chloroform (1/5th volume of trizol).
• Incubate for 2 to 5 minutes and centrifuge at 12000 rpm for 15 minutes at 4° C
• Transfer the aqueous phase into a new tube and add an equal volume of isopropanol. Mix well.
• Centrifuge at 10000 rpm for 10 minutes at 4° C.
• Discard the supernatant and resuspend the pellet in 70% ethanol.
• Again centrifuge at 10000 rpm for 10 minutes at 4° C.
• Discard the supernatant. Air dry the pellet at 37° C for 10-15 minutes
• Resuspend the pellet in 50 μL of TE buffer.
• Analyze the RNA sample quantitatively and qualitatively