RNA Extraction from Escherichia Coli.pptx

This simple laboratory PPT was designed for UPES-SOHST students as a guide for illustrating the experiment mentioned above, kindly share to help someone learn

1. RNA Extraction from Escherichia Coli 1 By: Okechukwu Francis Programme: PhD Biotechnology Supervisor: Dr. Nishu Goyal Date of Registration: SCHOOL OF HEALTH SCIENCE AND TECHNOLOGY ( SoHST )

2. Extracting RNA from Escherichia coli ( E. coli ) culture involves several critical steps to ensure the integrity and purity of the RNA. Cell Lysis is done to release cellular RNA and further RNA Precipitation to precipitate RNA molecules for final Extraction. 2 Principle Source: https://www.addgene.org/protocols/kit-free-rna-extraction/

3. E. coli Culture: Grown in an appropriate medium. TRIzol Reagent: A widely used reagent for RNA extraction. Chloroform: For phase separation. Isopropanol: For RNA precipitation. Ethanol (70% and 75%): For RNA washing and ethanol precipitation. DEPC-Treated Water: Diethyl pyrocarbonate -treated water for RNA resuspension. RNase Inhibitors: To protect RNA from degradation. Microcentrifuge Tubes: For sample handling. Centrifuge: For centrifugation steps. RNase-free Pipette Tips and Tubes: To prevent RNA degradation. Dry Ice or -80°C Freezer: For long-term RNA storage. Safety Gear: Lab coat, gloves, and safety goggles. 3 Materials and Reagents:

4. Cell Harvest (Preparatory stage): Grow E. coli culture to the desired density. Transfer the culture to a centrifuge tube. Centrifuge at 1,000 RPM for 2 minutes at 4°C to pellet the cells. Discard the supernatant. Cell Lysis: Resuspend the cell pellet in 1 mL of TRIzol reagent per 1 x 10^7 cells. Mix thoroughly and incubate at room temperature for 5 minutes. Phase Separation: Add 0.2 mL of chloroform to 0.8 mL of TRIzol used (1mL Trizol : Chloroform). Shake vigorously for 15 seconds. Incubate at room temperature for 2-3 minutes. Centrifuge at 5,000rpm for 5 minutes at 4°C. Transfer the aqueous phase (upper phase) to a new tube, avoiding the interphase. 4 Procedure:

5. RNA Precipitation: Add 0.5 mL of isopropanol per 1 mL of TRIzol gotten from step 3 aqueous phase. Mix gently by inverting the tube. Incubate at -20°C or -80°C for at least 1 hour to precipitate RNA. RNA Pellet Collection: Centrifuge at 2,000 RPM for 10 minutes at 4°C. Carefully remove the supernatant without disturbing the RNA pellet. Wash the RNA pellet with 1 mL of 75% ethanol. Centrifuge at 5000 RPM for 5 minutes at 4°C. Carefully remove the ethanol. RNA Resuspension: Air-dry the RNA pellet for 5-10 minutes at room temperature or under a fume hood. To resuspend the RNA pellet in 20-50 μL of DEPC-treated water. 5

6. RNase Treatment: Add 10 µL of RNase inhibitors to the RNA solution to protect it from degradation. Incubate at 37°C for 30 minutes to 1 hour to bind and inhibit any remaining RNase . RNA Storage: Store the RNA at -80°C or use it immediately for downstream applications. This methodology outline was designed for UPES- SoHST students as a guide for extracting RNA from an E. coli culture using TRIzol reagent. It's essential to work in a sterile environment and use RNase-free equipment to prevent RNA degradation. Adjust the procedure as needed for your specific experimental requirements and follow appropriate safety precautions in the laboratory. 6

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