Magnetic Bead-based Bacterial Total RNA Extraction Kit, 50 T (for Nucleic Acid Extraction)

Product Information

Description/Introduction

The kit is suitable for RNA extraction of Gram-positive bacteria and Gram-negative bacteria. Making use of the reversibly binding nucleic acid characteristics of superparamagnetic beads and carefully optimized lysis buffer system, the RNA in bacteria can be quickly and effectively separated. The extraction process can be completed in 30~40 min, and the RNA obtained has good integrity, high purity and high yield. It is suitable for RT-PCR, Real-Time PCR, Chip analysis, Northern Blot, Dot Blot, PolyA screening, In vitro translation, RNase protection analysis and molecular cloning and other downstream molecular biology experiments.

Storage and Handling Conditions

Lysozyme (50 mg/mL), DTT solution and DNase are shipped with wet ice and stored at -20°C. Other reagents are shipped and stored at room temperature; valid for 12 months.

Product Contents

Before starting (please read carefully)

1. Please bring your own 10 mM Tris-HCl (pH 8.0) for re-suspension of collected bacteria and dilution of lysozyme.

2. To extract RNA from Gram-positive bacteria, please prepare water bath or heating block at 37°C in advance.

3. If Buffer GRL precipitates, please heat it at 37°C and use it when it is restored to room temperature.

4. Please add DTT Solution to 4% to Buffer GRL before use, that is, 40 μL of DTT Solution for every 1 mL of Buffer GRL, preferably preparation as needed. Buffer GRL with DTT Solution can be stored at 4°C for a week.

5. Prepare only part of the DNase working solution required for the experiment.

6. Please add 18 mL anhydrous ethyl alcohol to Buffer RW1 before use, mix it thoroughly and then use it.

7. Please add 64 mL anhydrous ethyl alcohol to Buffer RW2 before use, mix it thoroughly and then use it.

Assay Protocol / Procedures

1. Harvest bacterial culture from 1~3 mL (the total amount of bacteria does not exceed 1×109) of bacterial fluid by centrifugation at 12,000 rpm (13,400 ×g) for 2 min at 4°C.

Note: The supernatant must be removed clean, otherwise it will affect the digestion of bacterial cell wall.

Note: Bacteria should not be excessive, otherwise it will cause problems such as yield reduction, genome residue and protein contamination. When OD600=1, it is recommended to take the amount of bacterial culture of 1 mL.

2. Suspend the bacterial body weight in 100 μL of 10 mM Tris-HCl (pH 8.0) containing lysozyme. The lysozyme concentration and incubation conditions are shown in the table below:

3. Add 200 μL of Buffer GRL (please confirm that DTT solution has been added before use) to the incubated bacterial suspension, and mix thoroughly with vortex oscillation.

4. Add 300 μL of anhydrous ethanol to the centrifuge tube and use a pipette to blow and mix thoroughly (precipitation may occur).

5. Add 20 μL of SweMag Beads to the mixture (SweMag Beads should be vortexed until evenly dispersed before use), use a pipette to blow until the magnetic beads are uniformly dispersed, and stand at room temperature for 5 min. During the period, use the pipette to blow and mix 2~3 times to keep the magnetic beads uniformly dispersed.

6. Move the centrifuge tube to the magnetic stand and let stand for 30 s, make the magnetic beads adsorb to the wall of the tube, when it is clear, absorb and discard the supernatant (in order to avoid affecting the extraction efficiency, do not suck the magnetic beads out), remove the centrifuge tube from the magnetic stand.

7. Add 500 μL of Buffer RW1 to the centrifuge tube, use a pipette to blow until the magnetic beads are dispersed evenly, then move the centrifuge tube to the magnetic stand and let stand for 30 s, make the magnetic beads adsorb to the tube wall, after it is clear, discard the supernatant (to avoid affecting the extraction efficiency, do not suck the magnetic beads out), remove the centrifuge tube from the magnetic stand.

8. Add 600 μL of Buffer RW2 to the centrifuge tube, blow it with a liquid pipette until the magnetic beads are dispersed evenly, then move the centrifuge tube to the magnetic stand for 30 s, make the magnetic beads adsorb to the tube wall, after it is clear, discard the supernatant (to avoid affecting the extraction efficiency, do not suck the magnetic beads out), remove the centrifuge tube from the magnetic stand.

9. Prepare DNase working solution: 85 μL Nuclease-free Water, 10 μL 10×DNase Buffer and 5 μL DNase were placed in a new Nuclease-free centrifuge tube, and gently mixed with a liquid pipette.

10. Add the DNase working solution to the centrifuge tube along the pipe wall, wash down the magnetic beads from the pipe wall and gently blow the mixture with a pipette. Stand at room temperaturet for 15 min, during which the pipette is used to blow and mix evenly several times.

11. Add 600 μL of Buffer RW2 to the centrifuge tube, blow it with a pipette until the magnetic beads are dispersed evenly, then move the centrifuge tube to the magnetic stand and let stand for 30 s, make the magnetic beads adsorb to the tube wall, and after being clear, discard the supernatant (do not suck the magnetic beads out to avoid affecting the extraction efficiency).

12. Repeat step 11.

13. Open the centrifuge tube lid, leave it at room temperature for 5~10 minutes to make the ethanol evaporate completely (Do not over-dry the beads as they may clump together and affect nucleic acid yield).

14. Remove the centrifuge tube from the magnetic stand, add 50~100 μL of Nuclease-free Water to the centrifuge tube, gently blow until the magnetic beads are uniformly dispersed with a pipette, stand at room temperature for 3~5 min, and use the pipette to mix 2~3 times.

15. Move the centrifuge tube to the magnetic stand until the magnetic beads are completely adsorbed, and absorb the supernatant into a new centrifuge tube to obtain high purity RNA.

16. The obtained RNA solution should be stored at -80°C.

Note

1. Please read the Product Manual carefully before use.

2. During the experiment, you should wear experimental clothes, disposable gloves and masks, avoid talking, and make sure that the utensils used in the extraction of RNA and the water or reagents used to prepare the solution are Nuclease-free products.

3. The degree of difficulty of enzymatic hydrolysis of the cell wall of different bacteria is different, and the cell wall of Gram-positive bacteria is generally more difficult to be hydrolyzed. Customers can adjust the concentration of lysozyme and incubation time according to the type of bacteria.

4. Some of the reagents are easy to volatilize and oxidize, so the reagents should not be exposed to the air for a long time, and the lid should be closed in time after using the reagents.

5. RNA extraction special operation test bench and electrophoresis equipment should be used.

For Research Use Only!

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