RNA Preparation - Bacterial RNA

Jeff Lawrence

1. Grow 2 mL culture overnight. Add 0.1 mL to 10 mL of fresh medium and grow to Klett 40 - 50 (OD600 0.2 - 0.3). Use more culture if a time course is taken.

2. Add 0.5 mL of M9 Medium, 20 mM Sodium Azide. Chill on ice. If doing a time course, freeze one aliquot and allow the flask to shake.

3. Spin at 10 krpm for 10 min in polypropylene centrifuge tubes.

4. Decant supernatant and resuspend pellet in 10 mL Protoplasting Buffer:

	1.5 mL	1 M Tris, pH 8.0
	15.4 g	Sucrose
	3.2 mL	250 mM EDTA, pH 8.0
	Adjust to 100 mL with ddH₂O

5. Add 80 µl of 50 mg/ml Lysozyme. Incubate on ice for 15 min.

6. Centrifuge at 7 krpm for 5 min. Decant supernatant and resuspend pellet in 0.5 mL Lysing Buffer:

	1 mL	1 M Tris, pH 8.0
	200 µL	5 M NaCl
	29 mg	Sodium Citrate
	7.5 µL	20% SDS
	91.3 µL	ddH₂O

7. Mix and transfer to a microfuge tube containing 15 µl DEPC. Incubate at 37° for 5 min.

8. Chill on ice. Add 250 µl Saturated 40% NaCl. Mix by inversion; incubate for 10 min on ice - a precipitate should form.

9. Centrifuge for 10 min. Divide supernatant between two clean tubes.

10. Phenol extract, chloroform extract, and precipitate each tube with 1 mL 95% ethanol.

11. Chill at -70° for 60 min. Centrifuge 10 min at 4° and decant supernatant.

12. Add 100 µl 70% ethanol. Centrifuge 3 min at room temperature.

13. Air dry pellet and resuspend in 100 µl DEPC-treated water (combined tubes).

14. Read OD260 of a 1:500 dilution. Calculate concentration as OD260 * 500 * 40 = mL/ml

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu