Universal Plant RNA Extraction Kit with DNase I(SpinColumn)
Packing Specification：
|
R2116-50
|
Universal Plant RNA Extraction Kit with DNase I(SpinColumn)
|
50T
|
CNY1290
|
|
R2116-200
|
Universal Plant RNA Extraction Kit with DNase I(SpinColumn)
|
200T
|
CNY4640
|
For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
The unique lysis buffer rapidly lyses cells and inactivates cellular RNases. After adjusting the binding conditions with ethanol, RNA is selectively adsorbed to the silica matrix membrane in the spin column under high chaotropic salt conditions. Residual DNA is directly digested on the column by DNase I. Through a series of rapid washing and centrifugation steps, deproteinization buffer and wash buffer remove impurities such as cellular metabolites and proteins. Finally, pure RNA is eluted from the silica matrix membrane with low-salt RNase-free H2O.
Product Features:
1.The silica matrix membrane in the spin column is made of high-quality special adsorption membrane, with minimal differences in adsorption capacity between columns.
2.No toxic reagents such as β-mercaptoethanol, phenol, or chloroform are used, and no ethanol precipitation steps are required.
3.Simple and efficient: Operation for a single sample can generally be completed within 40 minutes.
4.Equipped with on-column DNase I digestion, the obtained RNA is free of residual DNase and can be directly used in reverse transcription quantitative real-time PCR, next-generation sequencing, microarray, RACE, and other experiments.
5.Extremely wide applicability among similar products: Can extract RNA from plants including rice, corn, wheat, Arabidopsis thaliana, tomato, tobacco, and general polysaccharide/polyphenol-containing plants such as cotton and holly.
6.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 2.0 to 2.2, with no DNA residue. Suitable for direct use in quantitative real-time PCR, RT-PCR, microarray, next-generation sequencing, Northern blot, and various other experiments.
Experimental Procedures:
Note: Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle, mix thoroughly, and mark with a check in the box immediately after adding to avoid repeated addition!
1.Direct Grinding Method (Recommended)
a.Weigh 100 mg of fresh plant tissue, cut it into small pieces quickly, and place it into a mortar (frozen or liquid nitrogen-preserved samples can be directly weighed 100 mg and placed into a mortar). Add 1 mL of Lysis Buffer RPA and grind thoroughly into a homogenate at room temperature. Note: Grind quickly to ensure immediate and full contact between the tissue and Lysis Buffer RPA to inhibit RNase activity.
b.Transfer the lysate to a centrifuge tube, vortex vigorously for 15 seconds, and centrifuge at 13,000 rpm for 5-10 minutes to pellet undigested debris.
c.Transfer 480 μL of the lysate supernatant to a new centrifuge tube (more or all supernatant can be taken if it does not exceed the capacity of the RNA spin column to increase yield). Add absolute ethanol equal to 0.5 volumes of the supernatant. Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge.
d.Proceed directly to Step 3 of the experimental procedures.
2.Liquid Nitrogen Grinding Method
a.Transfer 500 μL of Lysis Buffer RPA to a 1.5 mL centrifuge tube.
b.Grind an appropriate amount of plant tissue into fine powder in liquid nitrogen, transfer 50 mg of the fine powder to the aforementioned centrifuge tube containing RPA, and vortex vigorously by hand for 20 seconds to fully lyse the sample.
c.Pipette to mix well to assist lysis or vortex vigorously until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds), which can shear DNA, reduce viscosity, and improve yield.
d.Centrifuge the lysate at 13,000 rpm for 5-10 minutes to pellet undigested debris.
e.Transfer the lysate supernatant to a new centrifuge tube (more supernatant can be taken if it does not exceed the capacity of the RNA spin column to increase yield). Add absolute ethanol equal to 0.5 volumes of the supernatant. Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge.
f.Proceed directly to Step 3 of the experimental procedures.
Note: The liquid nitrogen grinding method can be scaled up by doubling the reagents and sample amount if needed to increase yield (i.e., use 1 mL of Lysis Buffer RPA and 100 mg of sample).
Experimental Procedures (Continued)
3.Transfer the mixed solution (less than 720 μL each time, can be added in two portions) to a SpinRA column (place the spin column into a collection tube), centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid. Ensure that all liquid has passed through the membrane after centrifugation with no residue left on the membrane. If necessary, increase the centrifugal force and time.
4.Add 350 μL of Deproteinization Buffer RW1, incubate at room temperature for 1 minute, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid.
5.Preparation of DNase I Working Solution: Pipette 45 μL of DNase I Buffer and 5 μL of RNase-free DNase I into a centrifuge tube, and gently pipette to mix well to prepare the working solution (scale up the volume proportionally when processing multiple columns).
6.Add 50 μL of the DNase I working solution to the center of the SpinRA column. Incubate at room temperature (20-30°C) for 15 minutes. Note: Add the working solution directly to the center of the membrane; do not let it drip onto the O-ring or the inner wall of the column.
7.Add 350 μL of Deproteinization Buffer RW1 to the SpinRA column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the spin column back into the collection tube.
8.Add 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first!), centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid. Repeat this step with another 500 μL of Wash Buffer RW.
9.Place the SpinRA column back into the empty collection tube, centrifuge at 13,000 rpm for 2 minutes to remove as much wash buffer as possible, avoiding residual ethanol in the wash buffer that may inhibit downstream reactions.
10.Remove the SpinRA column, place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 30-50 μL of RNase-free water to the center of the adsorption membrane (heating the water in a 70-90°C water bath in advance can improve yield), incubate at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute.
If the expected RNA yield is >30 μg, add 30-50 μL of RNase-free water and repeat Step 10, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 10 for higher RNA concentration. Repeating the elution once results in a high-concentration RNA eluate. Combining two separate eluates increases the RNA yield by 15–30% compared to a single elution, but the concentration is lower. Users can choose according to their needs.
Note: If quantitative real-time PCR is not required and only regular reverse transcription or gene fragment cloning is performed, the on-column DNase digestion step can be omitted. Specifically, change "Add 350 μL of Deproteinization Buffer RW1" in Step 4 to "Add 700 μL of Deproteinization Buffer RW1", and omit Steps 5, 6, and 7.
