| Description | One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Application：high-fidelity PCR, point mutation, and gene cloning, etc. The amplified PCR products by DNA polymerase are blunt-ended and can be used directlyOne unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Application：high-fidelity PCR, point mutation, and gene cloning, etc. The amplified PCR products by DNA polymerase are blunt-ended and can be used directly for blunt-end cloning. Prior to be cloned into T vectors, the PCR products generated by the DNA polymerase must be dA-tailed by incubation with a conventional Taq DNA polymerase at 72℃ for 5-10 minutes.Definition of activity: One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74℃.Purity: free of DNA endonuclease and exonuclease, phosphatase, and RNAase.Enzyme storage buffer: 20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200µg/ml BSA and 50% (v/v) glycerol.Inactivation or inhibition: DNA polymerase can be deactivated by phenol chloroform extraction.Source：Recombinant hyperthermophilic archaeon Pyrococcus-like DNA polymerase expressed in E. coli.Enzyme storage buffer：20mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 200µg/ml BSA and 50% (v/v) glycerol.Inactivation or inhibition：DNA polymerase can be deactivated by phenol chloroform extraction.Precautions：Because PCR reaction is extremely sensitive, contamination must be avoided during preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1.Prepare PCR reactions:a.Thaw PCR components at room temperature and mix well prior to use. Keep the DNA polymerase on ice.b.Set up the PCR reaction on ice as follows: To amplify dsDNA 6kbComponentFinal ConcentrationVolumeFinal ConcentrationVolumeNuclease-free water-(36.5-x)µl-(29-y)µl10X Buffer (with Mg2+)1×5µl1×5µldNTP (2.5mM each)0.25mM each5µl0.5mM each10µlTemplate DNA10pg-1µg*xµl10pg-1µg*yµlPrimer mixtures (10µM each)0.2µM each1µl0.4µM each2µl DNA Polymerase2.5U/50µl1µl2.5U/50µl1µlTotal volume-50µl-50µlNote 1: When multiple reactions are required, prepare a master mix including all reagents except template and primer, and then dispense to different nuclease-free PCR tubes. Sometimes, the master mix can also include template and primer.Note 2: When amplifying fragments larger than 6kb, the amount of template should be increased appropriately, but too much template DNA can also easily lead to non-specific PCR amplification products. * The recommended amount of template varies for different types of DNA. In a reaction volume of 50µl, use 100 ng of Mammalian genomic DNA, 100 ng of E. coli genomic DNA, or 5-30 ng of Plasmid DNA. c.Mix the PCR reaction mixture by gentle vortex or pipetting. Centrifuge briefly to collect liquid at the bottom of the PCR tube. d.(Optional) When using a thermocycler without a heated lid, place a drop of mineral oil onto the top of PCR reaction mixture.2.Transfer the PCR reaction to a thermocycler and run thermocycling conditions as follows:StepTo amplify dsDNA 6kbCyclesInitial denaturation94℃ 3min94℃ 3min1Denaturation94℃ 30sec94℃ 30sec30Annealing55℃ 30sec55℃ 30secExtension68℃ 15s/kb68℃ 1min/kbFinal extension68℃ 10min68℃ 15min1Hold4℃ forever4℃ forever-Note 1: The extension temperature can also be set to 72℃, but the amplification will be slightly reduced. For DNA fragments difficult to amplify, denaturation at 92℃ and extension at 72℃ are recommended. Note 2: PCR running conditions should be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc.Note 3: The optimal extension time varies depending on the amplicon length. For amplification of DNA fragments shorter than 6kb, the recommended extension time is 15 seconds per kb (e.g. use 15 seconds to amplify 1kb fragment and 30 seconds to amplify 2kb fragment). When amplifying DNA fragments longer than 6kb, the recommended extension time is 1 minute per kb (e.g., use 5 minutes to amplify 5kb fragment). Note 4: For initial PCR, the number of cycles can be set to 35 to ensure that the expected PCR product can be amplified. The number of cycles for semi-quantitative or quantitative PCR analysis must be optimized appropriately so that the PCR reaction does not reach a plateau.FAQ:1.Few PCR products or no specific bands.a.It could be due to poor design of primers. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work normally but not your primers, redesign primers.b.DNA to be amplified may have a high GC content. High GC genes are relatively difficult to be amplified. In such a case, GC-rich buffer suitable for amplifying DNA with high GC content can be used, and PCR reaction parameters should be adjusted accordingly. Direct addition of 1-10% DMSO or 5-20% glycerol is also helpful for amplifying fragments with high GC content.c.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d.The presence of secondary structure in primers, primer dimers or short primers, may result in poor annealing of primers to the target sequence. In this case, try touch down or other methods for annealing. A gradual cooling from 65℃ to 55℃ or 50℃ is usually used to make annealing more efficiently. e.The annealing temperature needs to be optimized. If necessary, use a temperature gradient to determine the optimal annealing temperature for each template-primer pair combination. f.Insufficient extension time. The extension time can be extended 2-5 times from the recommended extension time, and can be set to 5 minutes per 1kb for fragments difficult to amplify. g.Insufficient denaturation. To amplify long DNA or high GC DNA fragment, the initial denaturation temperature can be adjusted to 95℃ for 1 min or even 95℃ for 2-4 min.h.Perform PCR reactions on a different thermal cycler to avoid possible problems with the instrument.i.Insufficient number of PCR cycles. Try more PCR cycles, but do not exceed 40 cycles. j.Insufficient amount of template. Add more DNA templates or try nested PCR or secondary PCR. Nested PCR is to design another pair of PCR primers inside the original PCR primers, and then conduct PCR amplification again with the diluted first PCR product as template. Instead, secondary PCR uses the same primers for second PCR amplification with the diluted PCR product as template. Nested PCR usually can remove the non-specific DNA amplification, but secondary PCR cannot.k.DNA sample contains substances that inhibit the PCR reactions. In such a case, template DNA can be purified using appropriate DNA purification methods such as column purification.l.Use high-purity primers.m.Use high-quality dNTP mixture.n.Increase the amount of DNA polymerase appropriately.o.When non-specific products are produced, increase the annealing temperature appropriately.p.Positive and negative controls are always recommended when optimizing PCR reactions.2.Occurrence of non-specific bands or DNA smear when examined by agarose gel electrophoresis.a.Increase the annealing temperature by 2-5℃.b.Reduce the amount of DNA template.c.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d.Reduce the amount of DNA polymerase appropriately.e.Reduce the extension time appropriately... Read More | Bovine pancreatic deoxyribonuclease is an endonuclease which splits phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding polynucleotides with free hydroxyl group at the 3' position and phosphate group at the 5' position. The average chain length of a limit digest is aBovine pancreatic deoxyribonuclease is an endonuclease which splits phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding polynucleotides with free hydroxyl group at the 3' position and phosphate group at the 5' position. The average chain length of a limit digest is a tetranucleotide.Used for the removal of DNA from protein samples. Deoxyribonuclease I from bovine pancreas has been used in a study to compare several procedures for reducing RNase contamination in preparations of DNase. Deoxyribonuclease I from bovine pancreas has also been used in a study to investigate the effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis... Read More | Inquire | MAP kinase substrate (MBP) is a peptide substrate for ERK 1 and ERK 2 MAP kinases. Sequence contains amino acids 95-98 of myelin basic protein (MBP) including Thr 97 within the Pro-X-Ser/Thr-Pro MAP kinase substrate consensus sequence phosphorylation site.MAP kinase substrate (MBP)is a peptide MAP kinase substrate (MBP) is a peptide substrate for ERK 1 and ERK 2 MAP kinases. Sequence contains amino acids 95-98 of myelin basic protein (MBP) including Thr 97 within the Pro-X-Ser/Thr-Pro MAP kinase substrate consensus sequence phosphorylation site.MAP kinase substrate (MBP)is a peptide substrate for ERK 1 and ERK 2 MAP kinases... Read More | This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groupsExtract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked andHomogenized samples, in their unique high salt state, RNA specifically binds to silicon matrix membranes, greatly reducingEffectively removing organic solvent contamination while removing protein contamination, resulting in higher purity and quality of RNA. bookThe product can quickly extract total RNA from various cells or tissues, and can process 30-50 mg of tissue or 5 × 10 ⁶ cells each time,Can handle multiple different samples simultaneously. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the residual DNA can be utilizedUsing DNase without RNase for digestion and removal on the column, the extracted RNA can be directly applied to RT-PCR Experiments such as Northern Blot, Dot Blot, and in vitro translation. U665516 Component 50 T Storage U665516A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. U665516B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. U665516C TRIzon Reagent 60 mL 2-8℃. Protect from light. U665516D TRIzon PaI™ 10 mL 2-8℃. Protect from light. U665516E Buffer RW1 40 mL RT U665516F Buffer RW2 (concentrate) 11 mL RT U665516G RNase-Free Water 10 mL RT U665516H Spin Columns RM with Collection Tubes 50 sets RT U665516I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTPreparation and important precautions before the experiment:1.To prevent RNase pollution, attention should be paid to the following aspects:1) RNase's plastic products and gun heads to avoid cross contamination.2) Prepare the solution using water without RNase.3) 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.3. If TRIzon Reagent is found to have precipitates before use, it can be dissolved in a water bath at 56 ℃ for a few minutes.Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Usage：1. Sample processing1a. Organization: 30-50 mg of tissue is thoroughly ground in liquid nitrogen and 1 mL of TRIzon Reagent is added, or 1 mL of TRIzon Reagent is added to the tissue sample and homogenized. Attention: The sample volume should not exceed 10% of the volume of TRIzon Reagent.2a. Single layer cell culture: Remove the culture medium and add an appropriate amount every 10 cm ² Add 1 mL of TRIzon Reagent.3a. Cell suspension: Collect cells by centrifugation. Add 1 mL of TRIzon Reagent to every 5 × 10 µ m cell.2. After adding TRIzon Reagent, repeatedly blow a few times to fully crack the sample. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Add 200 to every 1 mL of TRIzon Reagent µ LTRIzon PaI ™， Cover the tube tightly, vigorously shake for 15 seconds, and let it sit at room temperature for 2 minutes.4. Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 10 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer, and the upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Move the upper aqueous phase to a new RNase Free centrifuge tube (provided).5. Add an equal volume of 70% ethanol (prepared without RNase water) to the obtained aqueous solution, invert and mix well.6. Add all the solutions 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 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.8. Preparation of DNase I mixture: Take 52 µ LRNase Free Water, add 8 to it µ L 10 x Reaction Buffer and 20 µ L DNase I (1 U/ µ L) Mix well and prepare to a final volume of 80 µ The reaction solution of L.9. Directly add 80 µ L DNase I mixture to the adsorption column and incubate at 20-30 ℃ for 15 minutes.10. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.11. Add 500 to the adsorption column µ L 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 back into the collection tube.12. Repeat step 11.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 and thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (enzyme digestion,. )PCR, etc.14. 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 14 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 14... Read More |