Bacteria Total RNA Extraction Kit

Composition

Storage

Lysozyme can be transported at room temperature, please store at 2~8℃ after receiving the product.

All other reagents can be stored at room temperature for up to 2 years without showing any reduction in performance and would be stable more than 2 years if stored at 2-8℃

Introduction

This product is suitable for the total RNA purification from ≤2.0×10⁹ bacterial cultures. After the bacterial are broken by lysozyme, they are lysed by Buffer L and RNA is released. The filtrate containing RNA is added to the Spin Column, the RNA is bound to the Spin Column, and the dissolved proteins and PCR inhibitors are filtered out. The RNA was washed by two kinds of buffer and eluted with RNase-free Water, which can be used in various molecular biology experiments such as RT-PCR, Northern blot, Dot blot, mRNA separation and soon.

Equipment and Reagents to Be Supplied by User

1.      Absolute ethanol and 70% ethanol

2.      RNase-free 1.5 ml microcentrifuge tube

3.      Pipette and pipette tip (RNase-free pipette tip with filter element is recommended)

4.      Disposable gloves and protective supplies and paper towels

5.      Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)

6.      Vortexer

7.      A laboratory for RNAase-free

Preparation before use

1.        If the centrifuge has a refrigeration function, set the temperature to 25℃.

2.        Set the water bath temperature to 37℃.

3.        According to the number of bacterial samples extracted at one time (calculated by adding 100 µl lysozyme solution to each sample), prepare an appropriate amount of 100 mg/ml lysozyme solution: For example, to extract bacterial genomic DNA from 6 specimens, 65 mg dry lysozyme powder is said to be added to 650 µl RNase-free Water to prepare 650 µl lysozyme solution.

Note: Repeated freeze-thaw lysozyme solution will reduce its activity. If more lysozyme solution is prepared at one time, it should be divided into small portions and stored at -20℃. If there is any remaining lysozyme solution after thawing and use, it should be discarded and cannot be frozen again.

4.      Add absolute ethanol to Buffer WA and Buffer WBR according to the instructions on the label of the reagent bottle and tick the box on the label to mark "Ethanol added".

5.      Wear latex gloves and a mask during the entire RNA extraction process, as both saliva and skin contain RNase.

6.      If further experiments require complete DNA removal, please add DNase Ⅰ digestion steps.

Protocol

1.     Use 1.5 ml microcentrifuge tube to collect ≤2.0×10⁹ bacterial culture, add 100 µl RNase-free Water, vortex to fully suspend bacterial.

* 1.0×109 bacterial correspond to the number of bacteria in a bacterial culture with 1 ml OD600=1.

* Certain bivalent cations may inhibit lysozyme activity, and if the bacterial culture contains bivalent cations (such as MRS Culture, etc.), add a washing step after centrifugation to collect the bacterial: Add 1 ml distilled Water, centrifuge the bacterial at 12000 rpm for 30 sec after vortexing, discard the supernatant, and then add 100 µl RNase-free water to fully suspend the bacterial by vortex.

* Some special bacteria will change the pH value of the medium after culture (such as Lactobacillus), which will inhibit the activity of lysozyme, and need to be washed once after centrifuging and collecting bacterial, the method is the same as above.

* bacterial collection method: Centrifuge at 12000 rpm for 30 sec to collect the bacterial in 1~2 ml of the bacterial culture with OD600=1 and discard the medium.

2.     Add 100 µl lysozyme solution, vortex for about 15 sec and incubate at 37℃ for 30-60 min.

* Most bacterial have fully broken the cell walls after 30 min incubation, but some bacterial with thicker cell walls (e.g., Staphylococcus aureus) need to be treated for 1-2 hours to completely break their cell walls. Please adjust the incubate time according to the different types of bacteria.

3.     Flick the wall of the tube to suspend the bacterial, add 400 µl Buffer L, vortex until the bacterial are completely lyseed and the solution is transparent.

4.     Transfer all the lysate to the Filter Column, close the lid and centrifuge at 13000 rpm for 2 min.

* Do not omit this step, otherwise it may lead to blockage of the Spin Column in the following steps.

* If the lysate does not fully filter through the Filter Column, excessive bacterial use is indicated. At this time, 300 µl of the filtrate should be transferred to a clean 1.5 ml microcentrifuge tube, and then add 300 µl 70% ethanol to the 1.5 ml microcentrifuge tube to mix well, and then all the mixture should be added to the Spin Column, close the lid. Centrifuge at 13000 rpm for 1 min. Discard the Filter Column and residual filtrate and proceed directly to step 7.

5.      Discard the Filter Column, add 600 µl 70% ethanol into the filtrate and directly inject it with the pipette tip for 6-8 times to mix well. Transfer 600 µl of the mixture into the Spin Column, close the lid and centrifuge at 13000 rpm for 1 min.

* If there is precipitation after mixing with 70% ethanol, please add the precipitation together into the Spin Column.

6.     Discard the filtrate, place the Spin Column back into the 2 ml collection tube, transfer the remaining mixture to the Spin Column, close the lid and centrifuge at 13,000 rpm for 1 min.

* The filtrate does not need to be completely abandoned. To avoid contamination of the centrifuge by the filtrate adhering to the mouth of the centrifuge tube, the 2 ml collection tube can be inverted and slapped once on a paper towel.

7.     Discard the filtrate, place the Spin Column back into the 2 ml collection tube, add 500 µl Buffer WA to the Spin Column, close the lid and centrifuge at 13000 rpm for 1 min.

* Ensure that anhydrous ethanol has been added to the Buffer WA.

8.     Discard the filtrate, place the Spin Column back into the 2 ml collection tube, add 600 µl Buffer WBR to the Spin Column, close the lid and centrifuge at 13,000 rpm for 1 min.

* Ensure that anhydrous ethanol has been added to the Buffer WBR.

9.     Discard the filtrate, place the Spin Column back into the 2 ml collection tube, and centrifuge at 14,000 rpm for 1 min.

* If the centrifuge does not reach 14,000 rpm, centrifuge at maximum speed for 2 minutes.

* Do not omit this step, or the purified RNA may be mixed with ethanol, which may affect the subsequent RT-PCR effect.

10.  Discard 2 ml collection tube, place the Spin Column in a clean RNase-free 1.5 ml microcentrifuge tube, add 50-100 µl RNase-free Water into the center of the column membrane, close the lid, stand at room temperature for 1 minute, centrifuge at 13000 rpm for 1 minute.

* If the centrifuge does not have a leak-proof cover, please change the centrifuge condition to 8000 rpm for 1 minute to avoid damage to the centrifuge due to the tube cover falling off.

11.  Discard the Spin Column, and the eluted RNA can be immediately used in various molecular biology experiments or stored below - 70℃ for later use.