Spin DNA/RNA/Protein Kit-HYC517

Components

Storage

Store at room temperature (15–30 °C).

Required Reagents

β-mercaptoethanol (freshly opened or dedicated for RNA extraction), 70% ethanol (prepared with RNase-free water), absolute ethanol.

Instructions

Preparations

1. Guidelines for RNase contamination prevention:

• Use RNase-free plastic consumables and pipette tips to avoid cross-contamination.
• Bake glassware at 180 °C for 4 h before use; soak plasticware in 0.5 M NaOH for 10 min, rinse thoroughly with water, then autoclave.
• Prepare all solutions with RNase-free water.
• Wear disposable masks and gloves throughout the experiment, and replace gloves frequently.

2. Avoid repeated freeze-thaw cycles of samples, as this will compromise the quality of extracted DNA, RNA and protein. Samples suspended in Buffer RL can be stored at −70 °C for up to one month.
3. Add β-mercaptoethanol to Buffer RL prior to use at a ratio of 10 μL β-mercaptoethanol per 1 mL Buffer RL. Buffer RL supplemented with β-mercaptoethanol remains stable at ambient temperature for one month.
4. Add absolute ethanol to Buffer RW2, Buffer GW1 and Buffer GW2 as instructed on bottle labels before first use.
5. Inspect Buffer RL for crystals or precipitates before use. If present, fully redissolve the mixture in a 56 °C water bath.
6. Carry out all centrifugation steps at ambient temperature using a bench-top centrifuge.

Operating Protocol

1. Sample Preparation
2. Adherent cultured cells: Dissociate adherent cells to prepare cell suspension (maximum input: 1×10⁷ cells). Pellet cells and completely discard culture medium. Add 600 μL Buffer RL (confirm β-mercaptoethanol supplementation before use), and pipette repeatedly for thorough lysis.

Note: Complete removal of culture medium is mandatory; residual medium will interfere with lysis and subsequent nucleic acid purification.

1. Animal tissue: Use no more than 30 mg animal tissue. Grind tissue into fine powder in liquid nitrogen and resuspend in 600 μL Buffer RL (pre-added with β-mercaptoethanol), or directly add 600 μL pre-supplemented Buffer RL and homogenize fully.

Note: Incomplete homogenization reduces total RNA yield.

2. Centrifuge the lysate from Step 1 at 12,000 rpm (~13,400×g) for 3–5 min. Carefully transfer supernatant to Spin Column DM assembled with a collection tube. Centrifuge at 12,000 rpm for 30–60 sec and retain flow-through. Transfer Spin Column DM to a new 2 mL collection tube, store at ambient temperature or 4 °C for subsequent genomic DNA extraction.

Note: Ensure no liquid remains on the column membrane. Re-centrifuge if necessary until all liquid passes through the membrane.

Total RNA Extraction

3. Add an equal volume of 70% ethanol (prepared with RNase-free water) to the flow-through collected in Step 2, mix thoroughly.
4. Load the entire mixture onto Spin Column RM fitted with a collection tube; load in multiple aliquots if volume exceeds column capacity. Centrifuge at 12,000 rpm for 20 sec. Preserve flow-through in the collection tube for total protein extraction.
5. Transfer Spin Column RM to a new 2 mL collection tube. Add 700 μL Buffer RW1 to the column, centrifuge at 12,000 rpm for 20 sec, discard waste flow-through and reinstall the column.
6. Add 500 μL Buffer RW2 (verify anhydrous ethanol supplementation before use) to Spin Column RM, centrifuge at 12,000 rpm for 20 sec, discard waste flow-through and reinstall the column.
7. Repeat Step 6 once.
8. Centrifuge at 12,000 rpm for 2 min and discard waste liquid. Air-dry the spin column at ambient temperature for several minutes.

Note: This step eliminates residual ethanol on the column, which inhibits downstream enzymatic reactions (restriction digestion, PCR, etc.).

9. Place Spin Column RM into a new RNase-free 1.5 mL centrifuge tube. Pipette 30–50 μL RNase-Free Water directly onto the center of the silica membrane, incubate at ambient temperature for 2–5 min, then centrifuge at 12,000 rpm for 1 min to collect RNA eluate. Store purified RNA at −70 °C to prevent degradation.

Notes:

• The elution volume of RNase-Free Water must not be less than 30 μL; insufficient volume lowers RNA recovery yield.
• Repeat Step 9 with fresh 30–50 μL RNase-Free Water to increase total RNA yield.
• Reload collected RNA eluate back onto the column and repeat Step 9 to obtain higher RNA concentration.

Genomic DNA Extraction

10. Add 500 μL Buffer GW1 (confirm anhydrous ethanol supplementation before use) to Spin Column DM, centrifuge at 12,000 rpm for 20 sec, discard waste flow-through and reinstall the column.
11. Add 500 μL Buffer GW2 (verify anhydrous ethanol supplementation before use) to Spin Column DM, centrifuge at 12,000 rpm for 2 min, discard waste flow-through and reinstall the column.

Note: Repeat Step 11 if higher DNA purity is required.

12. Centrifuge at 12,000 rpm for 2 min and discard waste liquid. Air-dry Spin Column DM at ambient temperature for several minutes to fully remove residual ethanol.

Note: Residual ethanol will interfere with downstream enzymatic reactions (restriction digestion, PCR, etc.).

13. Transfer Spin Column DM to a new centrifuge tube. Pipette 100 μL Buffer GE onto the center of the silica membrane without touching the column wall, incubate at ambient temperature for 2–5 min, then centrifuge at 12,000 rpm for 2 min to collect DNA eluate. Store purified DNA at −20 °C.

Notes:

• The elution volume of Buffer GE must not be less than 100 μL; insufficient volume reduces DNA recovery yield.
• To boost total DNA yield, add fresh 100 μL Buffer GE to the column and repeat Step 13. To obtain higher DNA concentration, reload the DNA eluate from Step 13 onto the column and repeat Step 13.

Total Protein Extraction

14. Add an equal volume of Buffer PZ to the RNA flow-through collected in Step 4, mix thoroughly, and incubate at ambient temperature for 10–30 min.
15. Centrifuge at 12,000 rpm for 10 min, discard supernatant completely.
16. Add 500 μL 70% ethanol, centrifuge at 12,000 rpm for 1 min, aspirate supernatant to the maximum extent possible.
17. Air-dry the pellet at ambient temperature for several minutes.

Note: Complete ethanol removal is required; over-drying renders protein pellets difficult to dissolve, while incomplete drying leaves residual ethanol that disrupts sample loading for electrophoresis.

18. Resuspend pellet in 100 μL Buffer PLS to obtain total protein solution.

Notes:

• Protein samples dissolved in Buffer PLS are compatible with SDS-PAGE and Western Blot, but incompatible with Bradford protein quantification. For Bradford assay, dissolve pellets with 5% SDS or select an appropriate solubilization buffer based on downstream applications.
• Adjust the volume of protein solubilization buffer according to starting sample input and downstream experimental requirements.
• Purified protein can be stored at −20 °C for several months or at 2–8 °C for several days.

Sample Preparation for SDS-PAGE & Western Blot

19. Mix protein sample with protein Loading Buffer, denature at 95 °C for 5–10 min, then cool to ambient temperature.
20. Centrifuge at 12,000 rpm for 1 min, transfer supernatant for subsequent SDS-PAGE or Western Blot analysis.