| Product Description | 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 |
RT | |
Self 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 processing
1a 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 extraction
3. 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 extraction
10. 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 extraction
14. 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.
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