Bacterial Total RNA (plus) Extraction Kit

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25℃) in a dry environment and can be preserved for 12 months. Lysozyme contains a preservative, allowing for transportation at room temperature; however, for long-term storage, it should be stored at -20℃

3. Instructions for Using the Reagent Kit

3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.

3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).

3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, liquid nitrogen, chloroform, sterile deionized water, and EP tubes.

4.  Introduction to the Reagent Kit

This RNA purification kit provides a rapid and efficient purification solution for bacterial RNA, suitable for most bacterial tissues. The kit employs DNA removal technology to effectively eliminate genomic DNA, and the resulting RNA samples typically do not require on-column digestion, reducing the risk of RNA degradation.

The RNA Fast Purification Kit allows for the extraction of total bacterial RNA (including nuclear RNA and cytoplasmic RNA) within 1 hour. The extracted RNA can be directly used for RT-PCR, Northern blotting, etc. The entire purification process does not involve toxic reagents such as phenol-chloroform, making the RNA purification kit well-suited for various sample types.

5. Experimental Principles and Procedures

6. Extraction Process

Precautions before starting the experiment:

A. Before use, add the specified amount of absolute ethanol to Wash Buffer 1 as indicated on the reagent bottle label, and mark with a check to indicate the addition of absolute ethanol.

B. Elution Buffer is a 0.1x TE solution containing a minimal amount of EDTA. If EDTA may impact subsequent experiments, it is recommended to replace the Elution Buffer with sterile deionized water.

1. Sample Processing:

A. Gram-positive bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 rpm for 2 minutes, collect bacterial cells (1.53 ml), discard the supernatant, add 100μl Lysozyme (10mg/ml), thoroughly mix the bacterial cells, and incubate at room temperature for 15-30 minutes.

B. Gram-negative bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 rpm for 2 minutes, collect bacterial cells (1.53 ml), discard the supernatant, add 10μl Lysozyme (10mg/ml), thoroughly mix the bacterial cells, and incubate at room temperature for 3-5 minutes.

2.Add 400μl RNA Extraction Buffer I, vortex to mix thoroughly.

3. Transfer the lysate to a DNA clearance purification column, centrifuge at 13,000 rpm for 2 minutes, collect the filtrate (RNA is present in the filtrate).

4. Accurately estimate the volume of the filtrate, add 0.5 times the volume of absolute ethanol, mix thoroughly; if precipitation occurs, it does not affect subsequent experiments.

5. Add the obtained liquid to the RNA extraction purification column (approximately 650~700μl each time), centrifuge at more than 8,000 rpm for 1 minute, discard the collected waste liquid, and reinsert the collection tube into the RNA extraction purification column for the next step.

6. Repeat step 5, add the remaining liquid to the RNA extraction purification column, centrifuge at more than 8,000 rpm for 1 minute, discard the waste liquid and the collection tube.

7. Place the RNA extraction purification column into a new collection tube, add 600μl Inhibitor Removal Buffer, centrifuge at more than 8,000 rpm for 1 minute, discard the waste liquid, and reinsert the RNA extraction purification column into the tube for the next step.

8. Add 700μl Wash Buffer 1 to the RNA extraction purification column, centrifuge at 14,000 rpm (20,000×g) for 2 minutes, extend the centrifugation time appropriately to ensure the membrane is thoroughly dried.

(Note: Confirm that absolute ethanol has been added to Wash Buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so membrane drying is crucial. After centrifugation, ensure there is no ethanol present before elution. Discard the waste liquid and the collection tube.

After washing with Wash Buffer 1, the membrane on the RNA extraction purification column should only have a slight color. After centrifugation, carefully remove the RNA extraction purification column without touching the collection tube to ensure no ethanol interference.)

9. Place the RNA extraction purification column into a new centrifuge tube, drip 100 μl Elution Buffer onto the membrane, incubate at room temperature for 5 minutes (15°C~25°C), centrifuge at more than 8,000 rpm for 1 minute.

(Note: Eluting RNA with 50 μl Elution Buffer can increase RNA concentration but decrease total RNA yield.)

10. Repeat the previous step.

(Note: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.)