| Description | Inquire | Inquire | This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization step, which can significantly reduce the background and enhance the brightness of the target band. P665901Component20 TStorageP665901ASilver Stain Sensitizer (500×)2×1 mLRTP665901BSilver Stain Enhancer3 mLRTP665901CSilver Stain2×250 mLRTP665901DSilver Stain Developer4×125 mLRT Matters needing attention1. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% acetic acid) in advance.2. Please use deionized water and clean glass or plastic containers during operation, and wear disposable gloves for operation.The entire silver dyeing process needs to be carried out on a shaker, with a rotation speed of about 60 rpm.4. Self prepared ethanol and glacial acetic acid are required.Instructions for useThe dosage of each solution in the following operation steps takes the gel with a size of 8.5 × 5.5 cm and a thickness of 1.0 mm as an example. The gel is immersed in the solution completely, and is operated on a shaker, with a general dosage of 25 ml. For large gel, the dosage of each solution should be scaled up according to the gel volume. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: glacial acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% glacial acetic acid) in advance.1. Water washing: After electrophoresis is completed, wash the gel twice with ultrapure water for 5 minutes each time.2. Fixation: Fix the gel twice with 25 ml of fixative solution for 15 minutes each time.3. Elution: Wash the adhesive twice with eluent, each time for 5 minutes.4. Water washing: Wash the glue twice with ultrapure water, each time for 5 minutes.5. Sensitization: put the gel washed in the previous step into the silver dye sensitization working solution, incubate it accurately for 1 minute at room temperature, and then wash it with ultrapure water for three times, each time for 20 seconds. Preparation of silver staining sensitization working solution: Take 50 µ l Silver Stain Sensitivity (500 x) and add it to 25 ml of ultrapure water, mix well.6. Silver staining: discard ultrapure water and incubate gel in silver staining working solution for 30 minutes. Preparation of silver staining working solution: Take 25ml Silver Stain and add 50 µ l Silver Stain Enhanced to mix well.7. Water washing: Quickly wash the glue twice with ultrapure water, with each washing accurately controlled for 20 seconds.8. Development: Immerse the washed gel in the developer immediately and incubate it at room temperature for 2-3 minutes until the protein strip is clear. Preparation of developer: Take 25ml Silver Stain Developer and add 30 µ l Silver Stain Enhanced to mix well. Attention: Within 30 seconds of development, protein bands begin to appear and continue to develop for 2-3 minutes. If the protein band appears lighter, the development time can be appropriately extended to 5 minutes or more.9. Termination: After washing the developer on the gel with the termination solution, soak the gel in a new termination solution to react for 10 minutes.Experimental imagesSilver staining results of BSA protein samples after 10% SDS-PAGE gel electrophoresisThe molecular weight of BSA protein is about 66 kD, and the loading amounts from left to right are 50 ng, 10 ng, and 5 ng, respectively... Read More | Product content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligationProduct content Q665687Component100 TStorageQ665687AQuick T4 DNA Ligase (15 U/µL)100 µL-20℃. Avoid freeze/thaw cycle.Q665687B2×Quick Ligation Reaction Buffer5×200 µL-20℃. Avoid freeze/thaw cycle. Product IntroductionThe Quick Ligation Reaction Kit allows ligation of DNA sticky or flush ends in 5 minutes at room temperature (25°C). The kit contains Quick T4 DNA Ligase and 2×Quick Ligation Reaction Buffer optimized for fast and efficient DNA ligation.The ligation efficiency of Quick Ligation is equivalent to 1 hour of conventional ligation with T4 DNA Ligase. The Quick Ligation products can be used directly in routine bacterial transformation experiments.matters needing attention1. This kit enables most of the linkage reactions to reach the reaction endpoint within 5 minutes or less at 25°C. Increasing the reaction time will not enhance the reaction efficiency. If you use the rapid connection reaction after 1 hour, the conversion efficiency will be significantly reduced; if the rapid connection reaction at 25 ℃ overnight, the conversion efficiency will drop to 75%.2. 2×Quick Ligation Reaction Buffer contains ATP, which should be thawed on ice and mixed thoroughly before use. It is recommended to freeze the buffer in small tubes for the first time, so as to avoid repeated freezing and thawing, which will affect the efficiency of DNA ligation.3. Since T4 DNA Ligase contains glycerol, which is sticky and easy to hang on the wall, it is recommended to collect the liquid to the bottom of the tube by centrifugation for a short period of time before use, and the tip of the lance should not go too deep into the liquid surface when taking samples to avoid sticking to the tip of the lance and causing losses.4. If the quick ligation product is used for electrotransformation, the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation, and it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation.Usage1. The reaction solution was prepared according to the following system:*The amount of Insert DNA used: the molar ratio of Vector DNA and Insert DNA is generally 1:3-1:8, and the appropriate molar ratio of Vector DNA and Insert DNA can be selected according to the experimental situation.Calculation of DNA molar number: DNA molar number (nmol)=DNA mass (ng)/( 660daltons x number of inserted DNA bases bp).2. mix gently and centrifuge briefly. react at 25°C for 5 minutes.Note: The reaction time should not exceed 15 minutes, otherwise the connection efficiency will be reduced.3. Do not perform heat inactivation reactions. Centrifuge instantly and collect the solution from the wall to the bottom of the tube.Note: Heat inactivation significantly reduces transformation efficiency due to the presence of PEG in the buffer.4. After the reaction, store the DNA ligation product at 0-4℃, and then carry out transformation experiments; you can also store the DNA ligation product at -20℃.Note: When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.5. Heat shock the ligation product to transform 50 µl of receptor cells or take 1-2 µl of ligation product to electroshock transform 50 µl of receptor cells.Note: 1) When transforming by chemical method, do not add more than 10% of the volume of the receptor cell for the ligation product.(2) If the quick ligation product is used for electrotransformation, it is recommended to use a centrifugal column to purify the ligation product from DNA before electrotransformation because the PEG in the quick ligation reaction system will affect the efficiency of electrotransformation... Read More | This kit is suitable for extracting total RNA from fresh whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin). It can process up to 1.5 ml of whole blood and elute to obtain high-purity RNA with a molecular weight greater than 200 bp. Multiple samples can be This kit is suitable for extracting total RNA from fresh whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin). It can process up to 1.5 ml of whole blood and elute to obtain high-purity RNA with a molecular weight greater than 200 bp. Multiple samples can be completed simultaneously within 1 hour. This product does not require the ultra centrifugation step of CsCl purification and LiCl or ethanol precipitation. It does not contain toxic solvents such as phenol or chloroform. The purified RNA effectively removes enzyme inhibitors and pollutants such as heme and heparin. It can be directly used in various molecular biology routine experiments, such as RT-PCR, Northern Blot, Dot Blot, in vitro translation, and so on.Self prepared reagents: β- Mercaptoethanol, 70% ethanol (prepared with water without RNase), anhydrous ethanol. R666034 Component 50 T Storage R666034A Buffer RBL (10×) 60 mL RT R666034B Buffer RL 35 mL RT R666034C Buffer RW1 40 mL RT R666034D Buffer RW2 (concentrate) 11 mL RT R666034E RNase-Free Water 10 mL RT R666034F Spin Columns FL with Collection Tubes 50 sets RT R666034G Spin Columns RM with Collection Tubes 50 sets RT R666034H RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RT Preparation and important precautions before the experimentTo prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads 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 0.5M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution 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 freezing and thawing, otherwise it will affect the yield and quality of RNA extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be dissolved again in a 56 ℃ water bath. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1 ml Buffer RL µ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. This reagent kit cannot be used for RNA extraction from frozen blood samples with anticoagulants added.6.10 × Buffer RBL needs to be diluted 10 times with water without RNase before use, and then stored at 2-8 ℃ after dilution.7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase.8. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Operation steps1. Add 5 times the volume of 1 x Buffer RBL to fresh anticoagulant whole blood samples of 0.5-1.5 ml (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex or invert and mix well. Incubate on ice for 10-15 minutes, mix twice during the incubation process.Attention: During the incubation process, the cloudy suspension will become transparent, indicating that red blood cells have been lysed. If necessary, the incubation time can be extended to 20 minutes. 2. Centrifuge at 4 ℃, 2100 rpm (~400 × g) for 10 minutes, and carefully discard the supernatant.3. Add 2 times the volume of the blood sample to the above precipitate with 1 x Buffer RBL (please dilute 10 x Buffer RBL with RNase free water before use), gently vortex, and resuspend the precipitate thoroughly. 4. Centrifuge at 4 ℃ and 2100 rpm for 10 minutes, carefully and thoroughly remove the supernatant.Note: This step must completely remove the supernatant, otherwise it will affect the lysis and lead to a decrease in RNA production.5. Add Buffer RL to the precipitate (check if it has been added before use β- Mercaptoethanol, 0.5-1.5 ml of blood sample added to 600 µ L Buffer RL, or less than 0.5 ml of blood sample added to 350 µ L Buffer RL, mix well.6. Transfer the obtained liquid to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, collect the filtrate, and discard the filter column.7. Add 1 volume (600) to the obtained filtrate µ L or 350 µ l) Mix 70% ethanol (prepared without RNase water) well.Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.8. 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 at once, it can be transferred in multiple batches. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.9. Add 700 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 9 with the following steps.1) Add 350 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.2) Preparation of DNase I mixture: Take 70 µ Reaction Buffer and 10 µ L DNase I storage solution, gently mix and prepare to a final volume of 80 µ The reaction solution of L.Attention: The above system is configured according to our company's DNase I (D665537) reaction system. Please refer to the corresponding manual for other company products.1) Add 350 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.2) Preparation of DNase I mixture: Take 70 µ Reaction Buffer and 10 µ L DNase I storage solution, gently mix and prepare to a final volume of 80 µ The reaction solution of L.Attention: The above system is configured according to our company's DNase I (D665537) reaction system. Please refer to the corresponding manual for other company products.3) Add 80 µ l of the prepared DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.4) Add 350 to the adsorption column µ Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.10. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.11. Repeat step 10. 12. Centrifuge at 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 can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, 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 13 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 13... Read More |