| 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 | Purity>90% SDS-PAGE.Background:Luteinizing Hormone (LH) is a 42 kDa heterodimer belonging to the glycoprotein hormone family. It is composed of noncovalently linked glycosylated alpha and beta chains. The alpha subunit (CG alpha ) is also a component of Follicle-Stimulating Hormone (FSH), ThyroidPurity>90% SDS-PAGE.Background:Luteinizing Hormone (LH) is a 42 kDa heterodimer belonging to the glycoprotein hormone family. It is composed of noncovalently linked glycosylated alpha and beta chains. The alpha subunit (CG alpha ) is also a component of Follicle-Stimulating Hormone (FSH), Thyroid-Stimulating Hormone, and Chorionic Gonadotropin. The unique beta subunit confers the protein’s specific biological action and is responsible for the interaction with its receptor. The approximately 20 kDa human CG alpha subunit shares 73% and 72% amino acid (aa) sequence identity with the mouse and rat orthologs, respectively. The approximately 18 kDa human LH beta subunit shares 71% and 72% aa sequence identity with the mouse and rat orthologs, respectively. Multiple isoforms of LH exist due to differences in the post-translational glycosylation, sialylation, and sulphation modifications of its subunits. The composition, longevity, and activity of the different LH isoforms vary throughout a woman’s menstrual cycle and reproductive life cycle. LH is produced and secreted by the anterior pituitary gland. Its secretion is controlled by Gonadotropin-Releasing Hormone from the hypothalamus; however, LH secretion can also be stimulated by estradiol. LH works in concert with FSH to regulate female reproduction; FSH stimulates follicular growth and LH induces ovulation. LH also drives formation of the corpus luteum by promoting progesterone production. Additionally, LH has been suggested to stimulate the adrenal gland in postmenopausal women to induce secretion of sulfated DHEA, a precursor to androgens. In the testis, LH induces Leydig cell production of testosterone. Hypersecretion of LH has been shown to occur in women with polycystic ovary syndrome and is associated with an increased risk of infertility and miscarriage. Additionally, increased serum LH levels are associated with decreased cognition and have been implicated in the development and progression of Alzheimer’s disease. receptor into an A-frame... Read More | Purity＞97% by SDS-PAGE and HPLC analyses.FunctionPigment epithelium-derived factor (PEDF) is encoded by the SERPINF1 gene in humans and found in verebrates. It is a secreted phosphoglycoprotein that belongs to the clade F subfamily, serpin superfamily of proteinase inhibitors. The PEDF is a Purity＞97% by SDS-PAGE and HPLC analyses.FunctionPigment epithelium-derived factor (PEDF) is encoded by the SERPINF1 gene in humans and found in verebrates. It is a secreted phosphoglycoprotein that belongs to the clade F subfamily, serpin superfamily of proteinase inhibitors. The PEDF is a noninhibitory serpin with neurotrophic, anti-angiogenic, and anti-tumorigenic properties. It is synthesized as a 418 a.a. about 50kDa precursor that contains a 19 a.a. signal sequence and a 399 a.a. mature region that shows a pyroglutamate at Gln20. Like other serpins, it contains three β-sheets, 810 α-helices, and a C-terminal RCL (reactive center loop). Unlike other serpins with Ser protease inhibiting activity. PEDF has functions of inducing extensive neuronal differentiation in retinoblastoma cells, inhibiting of angiogenesis. As it does not undergo the S (stressed) to R (relaxed) conformational transition characteristic of active serpins, it exhibits no serine protease inhibitory activity. PEDF is researched as a therapeutic candidate for treatment of such conditions as choroidal neovascularization, heart disease, and cancer... Read More | Purity≥95% SDS-PAGE.Endotoxin level<0.1 EU/µgFunctionInhibits factor X (X(a)) directly and, in a Xa-dependent way, inhibits VIIa/tissue factor activity, presumably by forming a quaternary Xa/LACI/VIIa/TF complex. It possesses an antithrombotic action and also the ability to associate Purity≥95% SDS-PAGE.Endotoxin level<0.1 EU/µgFunctionInhibits factor X (X(a)) directly and, in a Xa-dependent way, inhibits VIIa/tissue factor activity, presumably by forming a quaternary Xa/LACI/VIIa/TF complex. It possesses an antithrombotic action and also the ability to associate with lipoproteins in plasma.Post-translationalO-glycosylated... Read More | Trypsin is a member of the serine protease family. Trypsin cleaves peptides on the C-terminal end of lysine and arginine amino acid residues. The pH optimum of trypsin is pH 7 - 10. The enzyme is inhibited by serine protease inhibitors, e.g. PMSF, and by metal chelating agents, e.g., EDTA. Trypsin is a member of the serine protease family. Trypsin cleaves peptides on the C-terminal end of lysine and arginine amino acid residues. The pH optimum of trypsin is pH 7 - 10. The enzyme is inhibited by serine protease inhibitors, e.g. PMSF, and by metal chelating agents, e.g., EDTA. Recombinant Human Trypsin is a genetically engineered protein expressed in E.coli and purified by high pressure liquid chromatography. There are no contaminating enzyme activities such as carboxypeptidase A and chymotrypsin. No protease inhibitors such as PMSF are contained in the preparation.Animal origin free:The use of recombinant Human Trypsin eliminates the risk of virus presence, and of any other potential adventitious agents found in animal pancreas-derived trypsin. Recombinant human trypsin:The amino acid sequence is the same as the Human Trypsin 2.Stable:A sterile recombinant human trypsin lyophilized eliminates the contamination risks and decreases the chance of activity loss in the process of transport and storage.High purity:(1) Recombinant human trypsin provides increased specificity and eliminates contaminating activities found in lower purity enzymes.(2) No other contaminating proteases such as chymotrypsin or carboxypeptidase A... Read More |