Yeast RNA Extraction Kit with Lytic Enzyme(SpinColumn)
Packing Specification：
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R2122N-50
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Yeast RNA Extraction Kit(SpinColumn)
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50T
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CNY1080
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R2122-50
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Yeast RNA Extraction Kit with Lytic Enzyme(SpinColumn)
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50T
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CNY1260
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R2122-200
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Yeast RNA Extraction Kit with Lytic Enzyme(SpinColumn)
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200T
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CNY4540
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
After yeast cells are treated with Lytic Enzyme to remove the cell wall, the unique lysis buffer/β-mercaptoethanol rapidly lyses the 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. 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 a special adsorption membrane imported from a world-renowned company. There is minimal difference in adsorption capacity between columns, ensuring good reproducibility. It overcomes the drawback of unstable membrane quality in domestic kits.
2.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
3.Fast and simple: Operation for a single sample can generally be completed within 20 minutes.
4.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 1.9 to 2.0, with almost no DNA residue. Suitable for RT-PCR, Northern blot, and various other experiments.
Experimental Procedures:
Notes
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!
Before operation, add β-mercaptoethanol to Lysis Buffer RLT to a final concentration of 1% (e.g., add 10 μL of β-mercaptoethanol to 1 mL of RLT). This lysis buffer is best prepared fresh. The prepared RLT can be stored at 4°C for one month.
Add 0.2% β-mercaptoethanol to the required volume of Buffer SE and set aside for later use.
1.Small-Scale Yeast Culture Cells
a.Collect 1 mL (approximately 10⁷ cells) of logarithmic-phase yeast culture into a 1.5 mL centrifuge tube. Centrifuge at 12,000 rpm for 30 seconds and aspirate the supernatant as much as possible.
b.Add 100 μL of Buffer SE (ensure β-mercaptoethanol has been added to a final concentration of 0.2%), gently pipette to resuspend the cells thoroughly. Add approximately 20 μL of Lytic Enzyme stock solution according to the yeast amount, invert to mix well. Incubate at 37°C for 15-30 minutes to digest the cell wall, inverting several times during incubation to assist digestion.
If cell wall lysis is insufficient resulting in low yield, increase the amount of Lytic Enzyme to improve the working concentration, extend the digestion time, or increase the temperature to 45°C. For yeasts not suitable for Lytic Enzyme digestion, other methods such as vortexing with 0.5 mm glass beads or repeated freeze-thaw cycles can be used.
c.Add 380 μL of Lysis Buffer RLT (ensure β-mercaptoethanol has been added to a final concentration of 1%), pipette to mix well, then vortex vigorously by hand for 20 seconds to fully lyse the cells.
Generally, no obvious clumps or insoluble substances should be visible after adding the lysis buffer and thorough vortexing. In rare cases where clumps or insoluble substances remain, centrifuge the lysate at 13,000 rpm for 3 minutes to pellet undigested debris or insoluble material. Transfer the entire supernatant to a new centrifuge tube and proceed to the next step.
d.Add 280 μL of 96-100% absolute ethanol. Immediately pipette to mix well; do not centrifuge.
e.Proceed to Step 3 of the experimental procedures.
2.Medium-Scale Yeast Culture Cells
a.Collect 2-3 mL (approximately 3×10⁷ cells) of logarithmic-phase yeast culture into a 1.5 mL centrifuge tube (for volumes exceeding 1.5 mL, collect cells in the same centrifuge tube in two batches). Centrifuge at 12,000 rpm for 30 seconds and aspirate the supernatant as much as possible.
b.Add 300 μL of Buffer SE (ensure β-mercaptoethanol has been added to a final concentration of 0.2%), gently pipette to resuspend the cells thoroughly. Add 50 μL of Lytic Enzyme stock solution according to the yeast amount, invert to mix well. Incubate at 37°C for 15-30 minutes to digest the cell wall, inverting several times during incubation to assist digestion.
If cell wall lysis is insufficient resulting in low yield, increase the amount of Lytic Enzyme to improve the working concentration, extend the digestion time, or increase the temperature to 45°C. For yeasts not suitable for Lytic Enzyme digestion, other methods such as vortexing with 0.5 mm glass beads or repeated freeze-thaw cycles can be used.
c.Centrifuge at 13,000 rpm for 1 minute and aspirate the supernatant as much as possible.
d.Add 350 μL of Lysis Buffer RLT (ensure β-mercaptoethanol has been added to a final concentration of 1%), pipette to mix well, then vortex vigorously by hand for 20 seconds to fully lyse the cells.
Generally, no obvious clumps or insoluble substances should be visible after adding the lysis buffer and thorough vortexing. In rare cases where clumps or insoluble substances remain, centrifuge the lysate at 13,000 rpm for 3 minutes to pellet undigested debris or insoluble material. Transfer the entire supernatant to a new centrifuge tube and proceed to the next step.
e.Add an equal volume of 70% ethanol (prepared with DEPC-treated water, approximately 350 μL). Immediately pipette to mix well; do not centrifuge.
f.Proceed to Step 3 of the experimental procedures.
Experimental Procedures (Continued)
3.Transfer the mixed solution to a SpinRA column (place the spin column into a collection tube), centrifuge at 13,000 rpm for 30-60 seconds, and discard the waste liquid.
4.Add 700 μL of Deproteinization Buffer RW1, incubate at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. If DNA residue is significant, incubate at room temperature for 5 minutes after adding RW1 before centrifugation.
5.Add 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Repeat this step with another 500 μL of Wash Buffer RW.
6.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.
7.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 7, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 7 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.
