| 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 amplification, point mutation, and gene cloning, etc. The amplified PCR products by Plus DNA polymerase are dA-tailed at 3&#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 amplification, point mutation, and gene cloning, etc. The amplified PCR products by Plus DNA polymerase are dA-tailed at 3’ ends and can be directly cloned into T vectors.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: Plus 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：Plus 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 Plus DNA polymerase on ice.b.Set up the PCR reaction on ice as follows:To amplify dsDNA 6kbReagentFinal 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 Plus DNA polymerase appropriately.e.Reduce the extension time appropriately... Read More | Arachis hypogaea lectin or Peanut Agglutinin (PNA) is isolated from peanuts and purified by affinity chromatography. The lectin has a molecular weight of 110 kDa and consists of four identical subunits of approximately 27 kDa each. PNA is a carbohydrate-free protein that displays specificity towardsArachis hypogaea lectin or Peanut Agglutinin (PNA) is isolated from peanuts and purified by affinity chromatography. The lectin has a molecular weight of 110 kDa and consists of four identical subunits of approximately 27 kDa each. PNA is a carbohydrate-free protein that displays specificity towards ?-D-Gal(1-3)-D-galNAc. It has potent anti-T activity and can be used to distinguish between human lymphocyte subsets. PNA has been used in tumour tissue determination for transitional mucosa malignancies. The lectin also agglutinates neuraminidase-treated human erythrocytes at < 0.1 µg/ml after trypsin treatment of cells and its activity is inhibited by lactose and galactose. PNA lectin is provided as a white to light yellow lyophilized powder from a buffer containing 10 mM NH4HCO3. The purity is determined by SDS-PAGE, which generates one band at 25-27 kDa.● Ultrapure quality ● Strong anti-T activity ● Sugar specificity: ?-D-Gal-(1-3)-D-GalNAc ● Agglutinates rabbit erythrocytes at < 0.1 µg/ml after trypsin treatment of the cells ● Lyophilized powderProbe in histochemistry and immuno-histochemistry;Human erythrocyte/lymphocyte studies... Read More | The Leuconostoc GPDH exhibits dual coenzyme specificity, namely NAD and NADP (Olive and Levy, Biochem., 6, 730 730, 1967). When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8 | Purity>95% (SDS-PAGE&HPLC) Endotoxin level<0.1 EU/µgFunctionMay regulate apoptosis, cell proliferation and cell differentiation. Binds beta-galactoside and a wide array of complex carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the dephosphorylation of Lyn Purity>95% (SDS-PAGE&HPLC) Endotoxin level<0.1 EU/µgFunctionMay regulate apoptosis, cell proliferation and cell differentiation. Binds beta-galactoside and a wide array of complex carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the dephosphorylation of Lyn kinase.Gal-1 is also engaged in many protein-protein interactions. Gal-1 plays a number of crucial roles in neuronal cell differentiation and survival in both the central and the peripheral nervous systems, and the establishment and maintenance of T-cell tolerance and homeostasis in vivo... Read More | Purity:>98%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism. It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. Purity:>98%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：Heme oxygenase (HMOX) is the rate limiting enzyme in heme catabolism. It cleaves heme to biliverdin, carbon monoxide, and iron. The biliverdin is subsequently converted to bilirubin by biliverdin reductase. The mechanism of HMOX is unique in that heme serves as the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O2. HMOX activity is highest in spleen where senescent erythrocytes are sequestered and destroyed. Two isoforms, HMOX1 and HMOX2, are expressed in most tissues. HMOX1 is an inducible enzyme in response to heme, heavy metals, oxidative stress, cytokines, and many drugs. Whereas HMOX2 displays a constitutive expression. HMOX1 is expressed mainly in spleen, liver, and kidney, and HMOX2 is prominently expressed in the brain and testes. The increased expression of HMOX1 levels is related to a variety of pathological states, where it functions as a cytoprotective molecule through its by products. HMOX1 also plays important roles in the regulation of cell proliferation, differentiation, and apoptosis... Read More |