| Description | Inquire | This reagent kit is suitable for simultaneously isolating and purifying genomic DNA, total RNA, and total protein from the same cell or tissue sample. This product does not require dividing the sample into three parts to extract DNA, RNA, and protein separately, nor does it require dividing the This reagent kit is suitable for simultaneously isolating and purifying genomic DNA, total RNA, and total protein from the same cell or tissue sample. This product does not require dividing the sample into three parts to extract DNA, RNA, and protein separately, nor does it require dividing the purified total nucleic acid into two parts before purifying DNA and RNA separately. Therefore, it can maximize the recovery of DNA, RNA, and protein, and can be used for the purification of nucleic acid and protein in small and rare samples. The purified DNA, RNA, and protein can be eluted separately and directly applied to various downstream molecular biology operations. This reagent kit does not contain toxic substances such as phenol and chloroform, and does not require ethanol precipitation. The operation is simple and fast. The extracted genomic DNA can be used for PCR, Real time PCR, SouthBlot, Dot Blot, comparative genomic hybridization (CGH), gene analysis, and SNP analysis; Total RNA can be applied in experiments such as RT-PCR, cDNA synthesis, Northern Blot, Dot Blot, and gene chips; Total protein can be applied in electrophoresis and Western Blot, among others. A665492 Component 50 T Storage A665492A Buffer RL 35 mL RT A665492B Buffer RW1 40 mL RT A665492C Buffer RW2 (concentrate) 11 mL RT A665492D RNase-Free Water 10 mL RT A665492E Buffer GW1 (concentrate) 13 mL RT A665492F Buffer GW2 (concentrate) 15 mL RT A665492G Buffer GE 15 mL RT A665492H Buffer PZ 60 mL RT A665492I Buffer PLS 15 mL RT A665492J Spin Columns DM with Collection Tubes 50 sets RT A665492K Spin Columns RM with Collection Tubes 50 sets RT A665492L Collection Tubes 100 EA RT A665492M RNase-Free Centrifuge Tubes (1.5 mL) 100 EA RTSelf prepared reagents:β- Mercaptoethanol (for newly opened or RNA extraction), 70% ethanol (prepared with water without RNase), and anhydrous ethanol.Preparation and important precautions before the experiment:To prevent RNase pollution, attention should be paid to the following aspects:1) Use plastic products and gun heads without RNase to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) The solution should be prepared using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The sample should avoid repeated freeze-thaw cycles, otherwise it will affect the quality of DNA, RNA, and protein extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.3. Please add Buffer RL before use β- Mercaptoethanol, 1 ml Buffer RL with 10 µ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.Before the first use, anhydrous ethanol should be added to Buffer RW2, Buffer GW1, and Buffer GW2 according to the instructions on the reagent bottle label.5. Before use, please check if there is any crystallization or precipitation in the Buffer RL. If there is any crystallization or precipitation, please dissolve it again in a 56 ℃ water bath.6. All centrifugation steps are performed using a desktop centrifuge at room temperature. Operation steps:1. Material processing1a The cells cultured on the wall should be first processed into cell suspension (maximum extraction amount of 107 cells), collected cells, discarded the culture medium, and added 600 cells µ L Buffer RL (check if it has been added before use) β- Mercaptoethanol), repeatedly blow and beat to fully decompose.Attention: It is necessary to discard the culture medium completely, otherwise it will affect the lysis and subsequent nucleic acid purification steps.1b Take no more than 30 mg of animal tissue, grind it into fine powder with liquid nitrogen, and add 600 µ Buffer RL (check if it has been added before use) β- Mercaptoethanol, or directly add 600 µ L Buffer RL (check if it has been added before use) β- Mercaptoethanol, homogenization treatment.Attention: The homogenate should be sufficient, otherwise it will affect RNA production.2. Centrifuge the solution obtained in the previous step at 12000 rpm (~13400 × g) for 3-5 minutes. Carefully add the supernatant to the spin columns DM that have been loaded into the collection tube. Centrifuge at 12000 rpm for 30-60 seconds and collect the filtrate. Place the adsorption column DM in a new 2 ml collection tube at room temperature or 4 ℃ for DNA extraction. Attention: Ensure that there is no liquid residue on the adsorption column, and if necessary, repeat centrifugation until all liquids pass through the membrane of the adsorption column. Total RNA extraction3. Add 1 volume of 70% ethanol (prepared without RNase water) to the filtrate obtained in step 2, and mix well.4. Add all the solution obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added completely at once, it can be transferred in stages. Centrifuge at 12000 rpm for 20 seconds and retain the liquid in the collection tube for protein extraction.5. Place the adsorption column RM into a new 2ml collection tube and add 700 to the adsorption column RM µ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM into the recovery manifold.6. Add 500 to the adsorption column RM µ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the 2 ml collection tube.7. Repeat step 6.Centrifuge at 8.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).9. Place the adsorption column RM in a new 1.5 ml centrifuge tube without RNase, and add 30-50 to the middle of the adsorption column RM µ Place RNase Free Water at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 9 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 9.Genomic DNA extraction10. Add 500 to the adsorption column DM µ Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column DM into the recovery tube.11. Add 500 to the adsorption column DM µ Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column DM into the recovery tube. Attention: To further improve DNA purity, repeat step 11.Centrifuge at 12.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column DM at room temperature for a few minutes to thoroughly dry the ethanol in the column. Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column DM in a new centrifuge tube and add 100 to the middle of the adsorption column DM by suspending it in the air µ L Buffer GE, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, collect DNA solution, and store DNA at -20 ℃.Attention:1) The volume of Buffer GE should not be less than 100 µ l. Small volume affects the recovery rate.2) If we want to increase DNA production, we will µ Add a new Buffer GE to the adsorption column and repeat step 13; If you want to increase the DNA concentration, you can add the DNA eluent obtained in step 13 back onto the adsorption column and repeat step 13.Protein extraction14. Add 1 volume of Buffer PZ to the RNA extraction effluent (i.e. the solution obtained in step 4), mix well, and let it stand at room temperature for 10-30 minutes.Centrifuge at 15.12000 rpm for 10 minutes and discard the supernatant.16. Add 500 µ Centrifuge at 12000 rpm for 1 minute with 70% ethanol, and try to absorb the supernatant as much as possible.17. Place the centrifuge tube at room temperature for a few minutes to dry the precipitate.Attention: The purpose of this step is to remove residual ethanol. Excessive drying can make protein precipitation difficult to dissolve, and incomplete drying of residual ethanol can affect protein loading.18. Add 100 µ L Buffer PLS to obtain protein solution.Attention:1) The protein samples obtained by dissolving with Buffer PLS are suitable for SDS-PAGE and Western Blot detection, but not for Bradford method for protein quantification. If Bradford method is needed for protein quantification, 5% SDS can be used to dissolve the protein, or suitable protein dissolution buffer can be selected based on downstream experiments.2) The amount of dissolved protein buffer added is determined based on the initial sample size and specific downstream test requirements.3) The dissolved protein can be stored at -20 ℃ for several months and at 2-8 ℃ for several days.If protein samples require SDS-PAGE electrophoresis, the following operations can be performed:19. Add protein loading buffer to the protein sample, denature at 95 ℃ for 5-10 minutes, and cool the sample to room temperature. Centrifuge at 20.12000 rpm for 1 minute, extract the supernatant for downstream SDS-PAGE or Western blot tests... Read More | Format:2-ComponentEnzyme:Horseradish peroxidase | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product content: M665559Component50 TStorageM665559ABuffer GTT15 mLRTM665559BBuffer GL15 mLRTM665559CBuffer GW1（concentrate）13 mLRTM665559DBuffer GW2（concentrate）15 mLRTM665559EBuffer GE15 mLRTM665559FProteinase K1.25 mLRTM665559GSpin CoLumns DM with CoLLection Tubes50 Product content: M665559Component50 TStorageM665559ABuffer GTT15 mLRTM665559BBuffer GL15 mLRTM665559CBuffer GW1（concentrate）13 mLRTM665559DBuffer GW2（concentrate）15 mLRTM665559EBuffer GE15 mLRTM665559FProteinase K1.25 mLRTM665559GSpin CoLumns DM with CoLLection Tubes50 EART Product Introduction:This reagent kit is suitable for extracting high-purity total DNA from fresh or frozen mouse or rat tails. The method provided by this reagent kit is simple and feasible, and the purification process does not require phenol or chloroform extraction. It can obtain DNA fragments up to 50 kb, and can also effectively recover fragments of 100 bp. This reagent kit uses a unique lysis solution to effectively lyse mouse tail samples. The optimized buffer system efficiently binds the DNA generated after the lysis of mouse tail to the silica matrix adsorption column, while other pollutants can flow through the membrane; Inhibitors of PCR and other enzymatic reactions can be effectively removed through a two-step washing process, followed by washing with low salt buffer or water to obtain high-purity DNA. The purified DNA can be directly used for downstream experiments such as enzyme digestion, PCR, ReaL Time PCR, library construction, Southern BLot, and molecular labeling.Self prepared reagent: anhydrous ethanol.Preparation and important precautions before the experiment:1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.Before the first use, anhydrous ethanol should be added to BufferGW1 and BufferGW2 according to the instructions on the reagent bottle label.3. Before use, please check if there is any crystallization or precipitation in the Buffer GL. If there is any crystallization or precipitation, please dissolve the Buffer GL again in a 56 ℃ water bath.Operation steps:1. Take a tail of a rat or two mice with a length of 0.4-0.6 cm, grind it into fine powder in liquid nitrogen or cut it into pieces and place it in a centrifuge tube (provided by oneself). Join 180 µ L Buffer GTT, shake and mix well. Note: Ensure that the starting quantity of the organization does not exceed the recommended range.2. Add 20 µ L Protein K, vortex oscillation, thoroughly mix.3. Place in a 56 ℃ water bath until the tissue solution is completely clear. Generally, digestion is required for 6-8 hours. During the incubation process, vortex oscillation is required to evenly disperse the sample. Note: 1) If there is still gel like substance after incubation and vortex oscillation, digest overnight or add 20 more if necessary µ L Protein K digestion will not affect subsequent operations. 2) To remove RNA, add 4 after completing the above steps µ L 100 mg/mL RNase A solution, shake well and let stand at room temperature for 5-10 minutes.4.12000 rpm (~13400 × g) for 1 minute to remove undigested tissues similar to mouse hair. Transfer the supernatant to a new centrifuge tube (provided by oneself).5. Add 200 µ L Buffer GL, vortex oscillation, thoroughly mixed. Join 200 µ L anhydrous ethanol, vortex and shake, thoroughly mix. Short centrifugation allows the solution on the tube wall to be collected to the bottom of the tube.Attention: 1) After adding Buffer GL and anhydrous ethanol, immediately vortex and shake to mix well.2) If multiple samples are operated together, Buffer GL and anhydrous ethanol can be mixed in equal proportions and added to the samples together.3) The addition of Buffer GL and anhydrous ethanol may produce white precipitates, which will not affect subsequent experiments.6. Add all the solutions obtained in step 5 to the adsorption column (Spin CoLumins DM) that has been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Note: To further improve DNA purity, repeat step 8.9.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).10. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high.2) Incubating at room temperature for 5 minutes before centrifugation can increase yield.3) Use an additional 50-200 µ Re washing with L Buffer GE or sterilized water can increase yield.4) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 10 back onto the adsorption membrane and repeat step 10; If the elution volume is less than 200 µ L. It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 µ g. Recommended 50 µ L Buffer GE or off... Read More |