SPINeasy® DNA/RNA/Protein All-In-One Kit | Manuel d'utilisation

Instruction Manual Research Use Only SPINeasy DNA/RNA/Protein All-In-One Kit For the isolation of DNA, RNA, and Protein from Single sample. Size: 50 & 5 preps Storage: 15-25 °C Cat. No.: 116544050 (50 PREPS) 116544000 (5 PREPS) Content Version: May 2026  Table of Contents 1. Introduction to SPINeasy DNA/RNA/Protein All-In-One Kit............... 3 2. Kit Components and User Supplied Materials .............................. 4 3. Storage and Kit Stability ...................................................... 5 4. Important Consideration Before Use ........................................ 5 5. Safety Precautions ............................................................. 6 6. Protocol .......................................................................... 7 7. Flow Chart ..................................................................... 10 8. Data ............................................................................ 12 9. Troubleshooting .............................................................. 14 10.Product Use Limitation & Warranty ....................................... 16 www.mpbio.com 2  SPINeasy DNA/RNA/Protein All-In- One Kit 1. Introduction to SPINeasy DNA/RNA/Protein All-In-One Kit SPINeasy DNA/RNA/Protein All-In-One Kit utilizes a convenient workflow and silicamembrane spin columns to isolate DNA, RNA and protein components from the same sample, without the use of toxic substances such as phenol and chloroform. The use of our specially formulated Lysis Buffer R and Lysing Matrix A in combination with FastPrep® Instruments from MP Biomedicals enables highly efficient lysis of tissue samples within seconds. DNA, RNA and proteins are then sequentially purified from the same lysate (Figure 1). Briefly, DNA is adsorbed onto the first spin column, while the flow-through is collected and used for RNA purification. The second spin column captures RNA and the flow-through from this step is used for protein extraction. DNA and RNA are bound on the first and second columns, respectively, and then washed and eluted. In the final extraction, proteins are precipitated out of solution, pelleted down by centrifugation, washed and resuspended. Each molecular component is then immediately available for their respective downstream applications. Visit www.mpbio.com to explore additional products to support your research. Kit Specifications at a Glance Technology Silica membrane technology Format Mini spin column Sample Tissues and Cell Cultures Sample amount 30 mg for tissues, 1x106 cells Elution volume 50–100 ?L for DNA/RNA 3 www.mpbio.com [Link] http://www.mpbio.com/ 2. Kit Components and User Supplied Materials 2.1 SPINeasy DNA/RNA/Protein All-In-One Kit Component 50 PREPS 5 PREPS ?Cat.No.?116544050? ?Cat.No.?116544000? Components Package Cat. No. Package Cat. No. Equilibration Buffer R 24 mL 116554063 2.5 mL 116554013 Lysing Matrix A 50 ea 116910050 5 ea 116910005 Lysis Buffer R 60 mL 116541052 6.0 mL 116541002 Wash Buffer D 30 mL 116544051 3.0 mL 116544001 Wash Buffer R 12 mL 116543051 1.2 mL 116543001 Elution Buffer GD 10 mL 116532054 1.0 mL 116532004 Nuclease-free water 10 mL 116541054 1.0 mL 116541004 DNase I 1 vial 116541055 1 vial 116541055 DNase I Buffer 5 mL 116541056 500 µL 116541006 Protein Precipitant 80 mL 116544052 8.0 mL 116544002 Column A with collection 100 ea 116544053 10 ea 116544003 tube Quick-start protocol 1 ea - 1 ea - Instruction Manual Available www.mpbio.com MSDS & CoA Available www.mpbio.com 2.2 User Supplied Materials • FastPrep Instrument - FastPrep-24TM 5G (Cat. No.116005500) • Vortex (an adapter for multiple microcentrifuge-sized tubes is recommended if multiple samples are to be processed simultaneously), if FastPrep Instrument is unavailable • Microcentrifuge capable of at least 14,000 x g • Absolute ethanol (at least 137.5 mL) • 50% Ethanol (at least 25 mL) • Nuclease-free 2 mL microcentrifuge tubes • Nuclease-free 1.5 mL microcentrifuge tubes • Buffer for resuspending protein pellet, such as SDS-PAGE loading buffer, 5% SDS, 50 mM NaOH/ 50 mM HCl (refer to Section 6.4) www.mpbio.com 4  SPINeasy DNA/RNA/Protein All-In- One Kit 3. Storage and Kit Stability DNase I should be stored at 2-8?C. All other components and reagents of SPINeasy DNA/RNA/Protein All-In-One Kit can be stored at room temperature (15-25?) until the expiration date printed on the kit label. For extended storage or storage in dry condition (humidity < 40%), store the Columns at 2-8?C to maintain performance. 4. Important Consideration Before Use ? Add 100 mL (10 mL for sample kit) of absolute ethanol to each bottle of Wash Buffer R and mark the bottle. ? Prepare DNase I solution according to instructions in Section 6.1. ? Prepare 750 µL of ethanol per prep for RNA binding. ? Prepare 500 µL of 50% ethanol per prep for washing of protein pellet. ? Prepare 100 – 200 µL of buffer of choice* per prep for resuspension of protein pellet. *Refer to note in Section 6.4. ? Centrifugationspeed stated in the manual will be a guideline; use the maximum speed available if 14,000 x g is not feasible. 5 www.mpbio.com  5. Safety Precautions Lysis Buffer R and Wash Buffer D contain components that can be harmful if swallowed and may cause irritation when in contact with skin and eyes. To prevent accidental ingestion, do not eat, drink or smoke when using this product. Wear personal protective equipment (gloves, lab coat and eye protection) to prevent contact with the skin or mucous membranes. Consult the Material Safety Data Sheet at www.mpbio.com for additional details. www.mpbio.com 6  SPINeasy DNA/RNA/Protein All-In- One Kit 6. Protocol 1. Preparation of DNase I Solution Briefly spin down the vial of lyophilized DNase I provided and resuspend with 500 µL of Nuclease-free water. Mix well to dissolve. Store DNase I solution at -20 °C in aliquots and avoid repeated freeze-thawing. Note: Do not prepare DNase I solution in DNase I buffer. 2. DNA Extraction Protocol 1. Tissues: Weigh 10 – 30 mg of tissue sample. Cut tissue into small pieces, transfer to a vial of Lysing Matrix A and add 1 mL Lysis Buffer R. Cell culture: Resuspend cell pellet (1 x 106 cells recommended) in 1 mL Lysis Buffer R and transfer to a vial of Lysing Matrix A. 2. Homogenize in a FastPrep Instrument for 15 seconds at speed setting of 4.0 m/s. If a FastPrep Instrument is not available, lysis may be performed by vortexing samples in Lysing Matrix A at the maximum speed for 3 – 5 mins. 3. Centrifuge at 14,000 x g for 10 mins. Note: Centrifuge at the maximum speed for all steps if 14,000 x g is not feasible. 4. Transfer the supernatant (~750 µL) to a Column A with collection tube. 5. Centrifuge at 14,000 x g for 1 min. 6. Transfer the flow-through to a clean 2 mL microcentrifuge tube and place the column back onto the collection tube. The flow-through will be used for RNA extraction (Section 6.3). 7. Add 500 µL of Wash Buffer D to the column. 8. Centrifuge at 14,000 x g for 1 min. Discard flow-through and reuse collection tube. 9. Add 500 µL of Wash Buffer R to the column. Incubate at room temperature for 1 min. 10. Centrifuge at 14,000 x g for 1 min. Discard flow-through and reuse collection tube. 11. Centrifuge at 14,000 x g for an additional 1 min to dry column. 12. Optional: Incubate at 55 °C for 3 – 5 mins to dry column completely. 13. Remove collection tube and place column onto a clean 1.5 mL microcentrifuge tube. 14. Add 100 µL of Elution Buffer GD to the center of the membrane. Incubate at room temperature for 1 min. For samples with low DNA content, reducing the elution volume to 50 µL may increase the concentration of eluted DNA. 15. Centrifuge at 8,000 x g for 1 – 2 mins to elute DNA. 7 www.mpbio.com  16. Eluted genomic DNA will be collected in the microcentrifuge tube. Store eluted DNA at -20 °C. 3. RNA Extraction Protocol 1. Add 750 µL of absolute ethanol to the flow-through collected in Step 6 of DNA Extraction Protocol (Section 6.2). Mix well by pipetting up and down. 2. Transfer 750 µL of the mixture to a new Column A with collection tube. 3. Centrifuge at 14,000 x g for 1 min. 4. Transfer the flow-through to a clean 2 mL microcentrifuge tube and place column back onto the collection tube. The flow-through will be used for protein extraction (Section 6.4). 5. Repeat steps 2 – 4 to load the remaining mixture and collect all the flow-through for protein extraction. 6. Add 500 µL of Wash Buffer R to the column. 7. Centrifuge at 14,000 x g for 1 min. Discard flow-through and reuse collection tube. 8. DNase I digestion: In a clean 1.5 mL microcentrifuge tube, add 5 µL of DNase I solution to 75 µL of DNase I Buffer per prep. Mix well and add 80 µL to the center of the column membrane. Incubate at room temperature for 15 mins. 9. Add 500 µL of Wash Buffer R to the column. 10. Centrifuge at 14,000 x g for 1 min. Discard flow-through and reuse collection tube. 11. Add 500 µL of Wash Buffer R to the column. 12. Centrifuge at 14,000 x g for 1 min. Discard flow-through and reuse collection tube. 13. Centrifuge at 14,000 x g for an additional 1 min to dry column. 14. Remove collection tube and place column onto a clean 1.5 mL microcentrifuge tube. 15. Add 100 µL of Nuclease-free water to the center of the membrane. Incubate at room temperature for 1 min. For samples with low RNA content, reducing the elution volume to 50 µL may increase the concentration of eluted RNA. 16. Centrifuge at 8,000 x g for 1 – 2 mins to elute RNA. 17. Eluted RNA will be collected in the microcentrifuge tube. For the best results, proceed to perform downstream applications immediately and keep RNA chilled on ice while working to prevent degradation. Store remaining RNA at -80 °C in aliquots and avoid repeated freeze-thawing. www.mpbio.com 8  SPINeasy DNA/RNA/Protein All-In- One Kit 4. Protein Extraction Protocol 1. Add an equal volume of Protein Precipitant to the flow-through collected in Step 4 of RNA Extraction Protocol (Section 6.3). 2. Mix well by inverting tubes several times and incubate at room temperature for at least 10 minutes to allow protein precipitation. 3. Centrifuge at 14,000 x g for 10 mins. Carefully discard supernatant. 4. Add 500 µL of 50% ethanol and invert tubes several times to wash pellet. 5. Centrifuge at 14,000 x g for 2 mins. 6. Carefully remove supernatant, leaving the protein pellet in the tube. 7. Dry pellet by uncapping the tube and leaving it at room temperature for 10 minutes or longer. 8. Resuspend pellet with 100 – 200 µL of the buffer of choice*, depending on the intended downstream application. *Note: For convenience, pellet may be dissolved in protein loading dyes, such as Laemmli buffer, and directly used for SDS-PAGE applications. Alternatively, pellet may be dissolved in 5% SDS solution. Note that SDS interferes with certain protein quantitation methods, such as the Bradford assay. Thus, buffers containing SDS, including protein loading buffers, are incompatible with such applications. If protein quantitation is necessary, the pellet may be dissolved in 50 mM NaOH, followed by neutralization with an equal volume of 50 mM HCl. 9 www.mpbio.com  7. Flow Chart www.mpbio.com 10  SPINeasy DNA/RNA/Protein All-In- One Kit 11 www.mpbio.com  8. Data The following results are examples of DNA, RNA and protein extracted from animal tissue samples using SPINeasy DNA/RNA/Protein All-In-One Kit. High yields and quality of genomic DNA and RNA are obtained, as indicated by A260/A280 and A260/A230 ratios, as the expected bands clearly resolved during agarose gel electrophoresis. Extracted DNA, RNA and proteins are suitable for downstream applications, such as PCR, RT-PCR and SDSPAGE , respectively. Table 1. Quantity and quality of DNA, RNA and protein extracted from animal tissues using SPINeasy DNA/RNA/Protein All-In-One Kit. Sample Starting Extraction Results Amount (mg) DNA RNA Protein Yield A260/280 A260/230 Yield A260/280 A260/230 Yield (µg/mg) (µg/mg) (µg/mg) Kidney 17.7 1.28 1.83 2.42 1.30 2.04 2.23 14.12 Spleen 12.4 1.81 1.84 2.45 1.81 2.04 2.22 20.81 Liver 17.0 1.09 1.84 2.89 3.48 2.07 2.17 10.16 Figure 1 (left). (A) DNA; (B) RNA; (C) Protein extracted from each animal tissue using SPINeasy DNA/RNA/Protein All-In-One Kit, analyzed using gel electrophoresis. M: DNA Marker; Lane 1: Kidney; Lane 2: Spleen; Lane 3: Liver. www.mpbio.com 12  SPINeasy DNA/RNA/Protein All-In- One Kit Figure 2: (A) PCR amplification of DNA and (B) RT-PCR amplification of RNA extracted from various samples using SPINeasy DNA/RNA/Protein All-In-One Kit. M: DNA Marker; Lane 1:Kidney; Lane 2: Spleen; Lane 3: Liver; Lane 4: Negative control. 13 www.mpbio.com [Link] mailto:apac-techsupport@mpbio.com 9. Troubleshooting This guide may be useful in solving any problems that may arise. For further assistance, please contact our technical support team at apac-techsupport@mpbio.com Problem Recommendation Low nucleic acid Yield / Ensure the extraction was performed according reduced integrity to kit manual instructions. Sample with low nucleic acid content: (i) Increase amount of starting material; (ii) Elute in a smaller volume (50 µL). Insufficient lysis or over-lysis: Adjust FastPrep speed and/ or duration to achieve the optimal homogenization condition for the sample type. When using a vortex instead of a FastPrep for sample homogenization, lysis condition may be optimized by testing reduced or extended vortexing duration. Poor sample quality. For best results, freshly prepared samples should be used. Poor elution: Ensure that Elution Buffer GD or Nuclease-free water is added to the center of the column membrane. RNase contamination. Work with nuclease-free tubes and pipette tips. Handle samples and perform all steps with clean gloves. Decontaminate work surfaces with RNase Erase® (Cat. No. 112440204). Smeared DNA/ RNA Poor sample quality. For best results, freshly Bands prepared samples should be used. Sample over-lysis. Reduce FastPrep speed and/or duration. Sample degradation: Use fresh samples or freshly frozen samples. It is recommended to store samples frozen in aliquots and avoid repeated freeze-thawing. RNA degradation. Work with freshly purified RNA and keep RNA chilled on ice after elution. RNA should be stored at -80 °C; avoid multiple freeze-thaw cycles. www.mpbio.com 14  SPINeasy DNA/RNA/Protein All-In- One Kit DNA Contamination in Perform on-column DNase I digestion according RNA Eluate to step 8 of the RNA extraction protocol (Section 6.3). Ensure DNase I solution is prepared according to manual instructions. Once dissolved, DNase I should be stored at -20 °C. No Protein Detected Protein pellet lost. After centrifugation, protein pellet may attach loosely to the side of the tube. Decant supernatant carefully to avoid losing the pellet. Protein Pellet Insoluble As every protein has a different isoelectric point, there will be proteins that are not soluble at a certain pH. Adjust the pH of the resuspension buffer to suit the protein of interest. Increase the volume of resuspension buffer to allow more proteins to dissolve. Smeared Protein Bands Sample overloaded. Dilute protein or reduce loading volume. Interference by insoluble material. After resuspending pellet, centrifuge briefly and use supernatant. 15 www.mpbio.com  10. Product Use Limitation & Warranty The products presented in this instruction manual are for research or manufacturing use only. They are not to be used as drugs or medical devices to diagnose, cure, mitigate, treat, or prevent diseases in humans or animals, either as part of an accepted course of therapy or in experimental clinical investigation. These products are not to be used as food, food additives or general household items. Purchase of MP Biomedicals products does not grant rights to reproduce, modify, or repackage the products or any derivative thereof to third parties. MP Biomedicals makes no warranty of any kind, expressed or implied, including merchantability or fitness for any particular purpose, except that the products sold will meet our specifications at the time of delivery. Buyer’s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited to, at our discretion, no replacement or compensation, product credits, refund of the purchase price of, or the replacement of materials that do not meet our specification. By acceptance of the product, Buyer indemnifies and holds MP Biomedicals harmless against, and assumes all liability for, the consequence of its use or misuse by the Buyer, its employees, or others, including, but not limited to, the cost of handling. Said refund or replacement is conditioned on Buyer notifying within thirty (30) days of receipt of product. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material(s). 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