| Description | The perfect KitAlysis Labware starter kit that combines the KitAlysis Inertion Box (Z742064) with the KitAlysis 24-Well Reaction Block and Screwdriver Set (Z742107).Provides an inert environment to run oxygen sensitive cross-coupling reactions in a laboratory fume hood.Designed to be used with The perfect KitAlysis Labware starter kit that combines the KitAlysis Inertion Box (Z742064) with the KitAlysis 24-Well Reaction Block and Screwdriver Set (Z742107).Provides an inert environment to run oxygen sensitive cross-coupling reactions in a laboratory fume hood.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:Ιnertion Box24-Well Reaction BlockTorque Screwdriver set with bitReaction Block Replacement Screws (10ea)... Read More | Inquire | Inquire | Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 EARTS665546ISpin Columns DM With Collection Tubes50 EARTProduct IntroductionThis kit provides a method for extracting total DNA from soil or fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples. It is also suitable for extracting DNA from samples containing high concentrations of PCR reaction inhibitors. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through washing steps. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as second-generation sequencing (16S amplicons and metagenomes), library construction, PCR, qPCR, Southern Blot, enzyme digestion molecular markers, etc.Self prepared reagents1. Constant temperature mixer - Product number: CW25932. Anhydrous ethanol, isopropanol3. Vortex oscillator or tissue grinderPreparation and important precautions before the experiment1. 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 Buffer GW1 (concentrate) and Buffer GW2 (concentrate) according to the instructions on the reagent bottle label.3. Take out the buffer RIL before use and store it at 2-8 ℃ immediately after use.Operation steps1. Centrifuge the Lysis Tube briefly to allow the beads to settle at the bottom.2. a. Add 0.1-0.3 g of soil or fecal sample to Lysis Tube, and add 740-820 µ L Buffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.b. If fecal samples are stored in non lytic fecal preservation solutions (such as CWY041S and CWY041M), add 200 to Lysis Tube µ L-600 µ L solid-liquid mixture, centrifuge at 13000 rpm for 1 minute, discard the storage solution (if the amount of solid after centrifugation is too small, it can be enriched again, but should not exceed 0.3g). Join 620 µ LBuffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.3. Fix the Lysis Tube in an oscillating grinding device equipped with a 2 mL adapter and process it according to the optimized grinding conditions of your equipment (see appendix).4. Shake the Lysis Tube on a constant temperature mixer at 70 ℃ and 1200 rpm for 10 minutes. Subsequently, centrifuge at 13000 rpm for 2 minutes to precipitate solid particles. Transfer 540 µ Transfer the supernatant to a new 2 mL centrifuge tube.5. Add 180 µ L Buffer RIL, vortex for 5 seconds, centrifuge at 13000 rpm for 2 minutes.Attention: Remove the buffer RIL before use and store it at 2-8 ℃ immediately after use.6. Add 160 to the new centrifuge tube in sequence µ L Buffer ML, 480 µ Supernatant from step 5, 320 µ L isopropanol, vortex for 5 seconds.7. Transfer the solution from the previous step to 650 µ Centrifuge at 12000 rpm (~13400 × g) for 1 minute into the spin columns DM that have been loaded into the collection tube.8. Discard the waste liquid in the collection pipe and place the adsorption column back into the collection pipe. Repeat step 7 until all the solution has been transferred.9. 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.10. 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. 11. Repeat step 10.12.12000 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 centrifuge tube (self provided) and add 50-200 drops of suspended droplets to the middle of the adsorption column µ L Buffer EBL 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) Incubating at room temperature for 5 minutes before centrifugation can increase yield.2) Use an additional 50-100 µ Further elution with L buffer or sterilized water can increase yield.3) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 13 back onto the adsorption membrane and repeat step 13, but it may reduce the total yield.4) The elution buffer does not contain chelating agents, please store DNA at -20 ℃.5) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can usually be resolved by diluting the DNA by 2-10 times.Appendix: Grind the sample using one of the following methods1. Manually vortex oscillate at maximum speed on the vortex oscillator for 10 minutes.2. On a vortex oscillator equipped with a 1.5-2 mL horizontal centrifuge tube holder, oscillate at maximum speed for 10 minutes (keeping the Lysis Tube horizontal). If the sample size exceeds 12, extend by 5-10 minutes. For example, using Scientific Industries or Mobile's Vortex Genie2 vortex oscillator.3.When using Qiagen's TissueLyser II, grind at 25Hz for 10 minutes.4.When using Qiagen's PowerLyzer 24 Homogenizer, homogenize at 2000 rpm for 30 seconds, pause for 30 seconds, and then homogenize again at 2000 rpm for 30 seconds.5.When using FastPrep-24 from MP Biomedicals, the recommended speed is 6.0 and the time is 40 seconds... Read More | This reagent kit is specially developed for one-step RT-PCR experiments. Reverse transcription and PCR are carried out in the same reaction system, without the need to add reagents or open the tube cap during the reaction process, which improves detection sensitivity and experimental efficiency This reagent kit is specially developed for one-step RT-PCR experiments. Reverse transcription and PCR are carried out in the same reaction system, without the need to add reagents or open the tube cap during the reaction process, which improves detection sensitivity and experimental efficiency while avoiding contamination. This kit includes a brand new high-efficiency reverse transcriptase, a fast hot start DNA polymerase, as well as reaction buffer suitable for reverse transcription and PCR amplification, and other components necessary for the experiment. The loss of activity of SuperRT reverse transcriptase RNase H reduces RNA degradation in reverse transcription reactions. This reverse enzyme has high reverse transcription efficiency and can perform good reverse transcription reactions on a small amount of RNA templates. The rapid hot start DNA polymerase used in PCR reaction has excellent performance of high amplification efficiency, strong specificity, and fast extension speed. The unique buffering system maximizes the efficiency of both reverse transcriptase and polymerase. The target product amplified using this reagent kit has an A base attached to the 3 'end, which can be directly used for T/A cloning.S665660Component100 TStorageS665660ASuperRT OneStep EnzymeMix50 µL-20℃. Avoid freeze/thaw cycle.S665660B2×SuperRT OneStep Buffer1.4 mL-20℃. Avoid freeze/thaw cycle.S665660CRNase-Free Water1.5 mL-20℃. Avoid freeze/thaw cycle. Notes:1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.3. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.4. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT-PCR reactions. When designing primers, factors such as GC content, primer length, primer position, and the secondary structure of PCR products need to be considered. It is recommended to use professional primer design software.Usage:1. Dissolve the RNA template, primers, OneStep RT-PCR Buffer, SuperRT OneStep RT-PCR EnzymeMix, and RNase Free Water and place them on ice for later use.2. Prepare the reaction system according to the following table: Reagent 25 µlReaction system Final concentration 2×SuperRT OneStep Buffer 12.5 µl 1× Forward Primer，10 µM 1 µl 0.4 µM Reverse Primer，10 µM 1 µl 0.4 µM SuperRT OneStep EnzymeMix 0.5 µl / RNA Template X µl 1 pg – 1 µg RNase-Free Water up to 25 µl / Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.3. Vortex and shake well, centrifuge briefly, and collect the solution to the bottom of the tube.4. Preheat the thermal cycler to 45 ℃, place the PCR tube in the thermal cycler, and perform RT-PCR reaction.Reaction conditions: Step Temperature Time / Reverse transcription 45℃ 30 min / PCR pre denaturation 95℃ 2 min Denaturation 94℃ 30 s 30-40 cycles Anneal 55-65℃ 30 s 30-40 cycles Extend 72℃ 30 s 30-40 cycles Finally extended 72℃ 5 min /Attention:1) In general PCR experiments, the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the annealing time is generally 20-30 seconds. If the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time is set based on the size of the amplified fragments, and the DNA Polymerase amplification efficiency contained in this product is 1 kb/30s.3) The number of cycles can be set based on the downstream application of the amplification product. Too few cycles, insufficient amplification; Multiple cycles increase the probability of mismatches and result in severe non-specific backgrounds. Therefore, while ensuring product yield, the number of cycles should be minimized as much as possible.5. After the reaction is complete, take 5 µ l of the reaction product, add an appropriate amount of loading buffer, and perform electrophoresis detection results... Read More |