| 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 | Inquire | Inquire | Purity>97% SDS-PAGE.FunctionReceptor for interleukin-2 | TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site.The substrate does not contain NMP (1-methyl2-pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal negative environmental impact.Product Characteristics TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site. The substrate does not contain NMP (1-methyl-2- pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal waste problems after use.Composition & Properties Ready-to-use substrate: Includes substrate buffer and hydrogen peroxide. No other reagents should be added.Working Procedure The following procedure is applicable to nitrocellulose membranes. The procedure must be optimized for other membranes.1.The desired amount of substrate is poured into a sealed container and allowed to reach room temperature, in the dark, before use. 2.After the last incubation with HRP-labelled Streptavidin or HRP-labelled secondary antibody it is recommended to wash the membrane in a 0.1 M Tris buffer pH 7.4.3.Shake off the excess buffer and incubate the membrane in the TMB-D Blotting solution for 10 minutes. 4.Wash the membrane in distilled water and allow it to dry. 5.The site of positive reaction will appear light blue with no or very little background staining.Tips & Tricks • The membrane can be blocked with Kementec’s Synthetic Blocking Buffer for Blotting, (cat. no. S494457). • For long-term preservation of the results, the membranes must be stored in the dark.Handling & Storage • Store solution at 2-8⁰C in the dark. • Avoid exposure to light, heat and contamination with metal ions or peroxidase. • Re-dispense only into bottles made of High-Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More |