RNApure Bacteria Kit（DNase I）

Item Number

R669890

• Storage Temp: Room temperature,Store at -20°C,Avoid repeated freezing and thawing
• Shipped In: Ice chest + Ice pads

Product Description

Products 

R669890	Component	50 T	Storage
R669890A	DNase I	1000 U	-20℃. Avoid freeze/thaw cycle.
R669890B	10×Reaction Buffer	1mL	-20℃. Avoid freeze/thaw cycle.
R669890C	Buffer RL	35 mL	RT
R669890D	Buffer RW1	40 mL	RT
R669890E	Buffer RW2 (concentrate)	11 mL	RT
R669890F	RNase-Free Water	10 mL	RT
R669890G	Spin Columns FL with Collection Tubes	50 sets	RT
R669890H	Spin Columns RM with Collection Tubes	50 sets	RT
R669890I	RNase-Free Centrifuge Tubes (1.5 mL)	100 EA	RT

Products

This kit adopts centrifugal adsorption columns with high efficiency and specificbinding of nucleic acids and unique buffer system, which can rapidly extract totalRNA from bacteria or cultured animal cells.The reaction can be completed in 30-40minutes, and the extracted total RNA is extremely pure and free of protein and othercontaminants, which is suitable for RT-PCR, Real-Time RT-PCR, microarray analysis,in vitro translation and other experiments. Self-contained reagents: Lysozyme, β-mercaptoethanol, anhydrous ethanol (freshlyopened or for RNA extraction). 

Pre-experiment Preparation and Important Notes 

1. To prevent RNase contamination, attention should be paid to the following aspects:

1) Use RNase-free plastics and tips to avoid cross-contamination. 

2) RNase-free water should be used to prepare the solution. 

3) Operators wear disposable masks and gloves, and change gloves diligently duringthe experiment.

2. Add β-mercaptoethanol to Buffer RL before use to reach a final concentrationof 1%, e.g., add 10 μl of β-mercaptoethanol to 1 ml of Buffer RL. Buffer RL withβ-mercaptoethanol can be stored at 4℃ for 1 month, if precipitation occurs, pleaseheat to dissolve and use.

3. Anhydrous ethanol should be added to Buffer RW2 before first use according tothe instructions on the reagent bottle label. 

4. All centrifugation steps are carried out at room temperature if not otherwisespecified, and all steps should be performed quickly.  

Procedure 

1. Centrifuge at 12,000 rpm (~13,400 x g) at 4°C for 2 minutes to collect theorganisms (maximum volume of organisms should not exceed 1 x 109) and carefullyremove all supernatants. Note: Supernatants that leave residues can interfere with the subsequent digestionprocess. 

2. Thoroughly resuspend the organisms with 100 μl of TE buffer containing Lysozymeand incubate at room temperature. The specific formulation and incubation time areas follows:

/	The final concentration of Lysozyme in TE buffer	incubation time
G-germ	400μg/ml	3-5min
G+germ	3mg/ml	5-10min

3. Add 350 μl of Buffer RL (check that β-mercaptoethanol has been added beforeuse), vortex and shake to mix (insoluble precipitate may appear in this step), addall of the solution and the precipitate to the filter columns (Spin Columns FL) thathave been loaded into the collection tubes, and centrifuge at 12,000 rpm for 2minutes. 4. Add 250 μl of anhydrous ethanol to the filtrate obtained in the previous stepand mix well (a precipitate may appear at this point). Transfer the resulting solution together with the precipitate to a Spin Columns RM packed in a collectiontube, centrifuge at 12,000 rpm for 1 min, discard the waste solution and put thecolumn back into the collection tube.

5. Add 350 μl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for1min, discard the waste liquid and put the adsorbent column back into the collectiontube.

6. Preparation of DNase I mixture: Take 52μl of RNase-Free Water, add 8μl of 10×Reaction Buffer and 20μl of DNase I (1U/μl) to it, mix well, and make a finalvolume of 80μl of reaction solution.

7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at20-30°C for 15 minutes.

8. Add 350 μl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for1min, discard the waste liquid and put the adsorbent column back into the collectiontube.

9. Add 500 μl of Buffer RW2 to the column (check that anhydrous ethanol is addedbefore use), centrifuge at 12,000 rpm for 1 min, and discard the waste solution.

10. Repeat step 9.

11. Place the adsorbent column back into the collection tube and centrifuge at 12,000rpm for 2 minutes. Note: The purpose of this step is to remove residual ethanol from the adsorptioncolumn; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).

12. Load the adsorption column into a new RNase-Free collection tube, add 30-50 μl of RNase-Free Water to the middle of the adsorption membrane, leave it at roomtemperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, collect the RNAsolution, and store the RNA at -70°C to prevent degradation. Note: 1) The volume of RNase-Free Water should not be less than 30 μl, too smallvolume affects the recovery rate. 2) If you want to increase the RNA yield, repeat step 12 with 30-50 μl of freshRNase-Free Water. If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated.