BioVision’s EasyRNATM Bacterial RNA Mini Kit provides a quick and easy method to isolate total RNA from total RNA of Gram-negative (B. subtilis) or Gram-negative (E. coli) bacteria in 30 minutes. Only traces of genomic DNA exist in purified RNA, which can be eliminated by treatment with DNase I (See details in the protocol) when necessary.
II. Sample type: for fast and efficient isolation of total RNA from plant tissues in 30 min.
User-supplied reagents and equipment:
• Benchtop microcentrifuge and sterile 1.5 mL tubes; Vacuum manifold if the vacuum protocol is used; 100% ethanol; βmercaptoethanol; Optional: DNase I, DNase buffer
V. Shipping and storage:
All reagents are shipped at room temperature. DNase I (optional) and lysozyme should be stored at -20 ° C. All other ingredients
can be stored at room temperature. All kit components are warranted for 12 months from the date of purchase.
SAW. Reagent preparation and storage conditions:
• Prepare all components and prepare all necessary materials by reviewing the protocol and becoming familiar with each step.
• Add 1% volume of β-mercaptoethanol to Buffer LY before use and store at 4oC.
• Add 80 ml (K1351-50) or 96 ml (K1351-250) of 100% ethanol to the RNA wash buffer before use.
• Add 9.6 ml (K1351-50) or 48 ml (K1351-250) of 100% ethanol to the DNase stop buffer before use. The final ethanol is 80% (v / v).
• Prepare a 3 mg/ml or 0.4 mg/ml lysozyme stock solution with elution buffer or TE buffer and distribute aliquots in suitable portions.
Store each aliquot at -20 ℃ and thaw before use.
VII. Protocol for the extraction of total RNA from Gram positive (B. subtilis) or Gram negative (E. coli) bacteria
1. Grow a bacterial culture overnight in the appropriate medium at a suitable temperature. The next day dilute the culture 1:50
with medium and grow until OD600 reads 0.6-1.0. This should only take a few hours. If growth is too slow, reduce dilution.
factor. Do not use overnight culture for RNA isolation!
2. Do not collect more than 3 mL of culture (<5×108
) by centrifugation at 3000 rpm for 10 min.
3. Gently remove the supernatant as much as possible.
4. Resuspend the pellet in 100 µL of fresh TE buffer (10 mM, TrisHCl, pH 8.0; 1 mM EDTA, pH 8.0) or elution buffer (10 mM
Tris-HCl, pH 8.5) containing lysozyme. (Use 3 mg of lysozyme per 1 ml of TE buffer for gram-positive bacteria and 0.4 mg / ml of lysozyme for
Gram-negative bacteria). Mix by tapping.
5. Incubate the resuspended pellet at room temperature for 5-10 minutes for Gram-positive bacteria, or 3-5 minutes for Gram-negative bacteria.
6. Add 400 µL of LY buffer. Mix gently. Centrifugation at 13,000 rpm for 5 min, transfer the clear lysate to a new RNase-free tube. Add 0.5
volume of 100% ethanol to the lysate (for example: 250 µL of 100% ethanol for 500 µL of lysate). Make sure that the β-mercaptoethanol has been
added before use.
7. Transfer the solution to the RNA column and centrifuge at 12,000 rpm for 1 min. Discard the collection tube with the flow
and replace the column in a new collection tube.
8. Add 500 µL of Buffer RB to the column and centrifuge at 13,000 rpm for 30 s. Discard continuous flow.
9. Add another 500 µl of RNA wash buffer to the column and centrifuge at 13,000 rpm for 30 s. Discard continuous flow. and put the
column back to the collection tube.
10. Centrifuge the column at 12,000 rpm, with the lid open, for an additional 1 min. Removal of residual ethanol is critical for optimal elution.
12. Place the column in a 1.5 ml RNase-free tube, add 50-100 µL of DEPC-treated ddH2O to the column and centrifuge at 13,000 rpm for
2 minutes. The RNA is in the flowing liquid. Store the RNA solution at -20 ℃.
Note: It is strongly recommended that the quality of the RNA be determined before subsequent applications. RNA quality can be assessed
by denatured agarose gel electrophoresis with ethidium bromide staining. Several sharp bands should appear in the gel, including
28S and 18S ribosomal RNA bands, as well as certain populations of mRNA and bands. If these bands extend towards a lower molecular weight
RNA weight, then the RNA has undergone significant degradation during preparation, handling or storage, RNA molecule less than 200
bases in length do not bind efficiently to the RNA column. An A260 / A280 ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid.