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 unique lysis buffer system combined with silica gel column purification technology, bacterial RNA can be extracted quickly and effectively, and the extraction process can be completed in 30~40 min. The RNA obtained has 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 a 37°C water bath or heating block in advance.

3. If Buffer BRL 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 DTT Solution for every 1 mL Buffer BRL, 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 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 liquid of 1 mL.

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

3. Add 350 μL Buffer BRL to the incubated bacterial suspension (please confirm that DTT solution has been added before use), and mix thoroughly with vortex oscillation.

4. Add 250 μL anhydrous ethanol to the centrifuge tube and use a pipette to blow and mix thoroughly (precipitation may occur). Transfer all the mixture and precipitation into RNA Spin Column, centrifuge at 12,000 rpm (~13,400×g) for 1 min at room temperature, and discard the filtrate.

5. Add 350 μL Buffer RW1 to RNA Spin Column and centrifuge at 12,000 rpm (~13,400×g) for 1 min at room temperature, then discard the filtrate.

6. Prepare DNase working solution: 40 μL Nuclease-free Water, 5 μL 10×DNase Buffer and 5 μL DNase were placed in a new Nuclease-free centrifuge tube, and gently mixed with a liquid pipette.

7. Add the DNase working solution droplet to the center of the membrane of the RNA Spin Column and stand at room temperature for 15 minutes.

8. Add 350 μL Buffer RW1 to RNA Spin Column, centrifuge at 12,000 rpm (~13,400×g) for 1 min at room temperature, and discard the filtrate.

9. Add 600 μL Buffer RW2 to RNA Spin Column (please add Buffer RW2 along the pipe wall to help flush the residual salt on the pipe wall), centrifuge at 12,000 rpm (~13,400×g) for 1 min at room temperature, and discard the filtrate.

10. Repeat step 9.

11. Put Spin Column on Collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 min at room temperature. Transfer Spin Column to a new Nuclease-free 1.5 mL centrifuge tube.

12. Open the lid for 3~5 min at room temperature, so that the residual ethanol of Spin Column can be evaporate completely.

13. Add 50~100 μL Nuclease-free Water to the centrifuge tube, stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min at room temperature to collect RNA solution. To get a higher concentration of RNA, you can also add the first eluent back to RNA Spin Column, then stand at room temperature for 5 min and centrifuge for 2 min to collect RNA again.

14. 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 extracted RNA should use Nuclease-free glass or plastic utensils in the process of continuing processing. Glassware can be baked in an oven at 150°C for 4 h, plastic ware can be soaked in 0.5M NaOH solution for 10 min. Then rinse with water and sterilize 30 min at 121°C to remove RNase.

4. 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.

5. 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.

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

For Research Use Only!

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