| Description | Aladdin's EnzymoPure™ High-Fidelity DNA Polymerase is a recombinant enzyme expressed in E. coli and purified using Aladdin's PerfectProtein™ technology platform. It is a modified thermostable DNA polymerase with a mutation in the uracil-binding pocket that enables the ability to read andAladdin's EnzymoPure™ High-Fidelity DNA Polymerase is a recombinant enzyme expressed in E. coli and purified using Aladdin's PerfectProtein™ technology platform. It is a modified thermostable DNA polymerase with a mutation in the uracil-binding pocket that enables the ability to read and amplify DNA templates containing uracil and hypoxanthine bases. Its strong 3´→5´ exonuclease (proofreading) activity results in superior accuracy during DNA amplification.The EnzymoPure™ High-Fidelity DNA Polymerase generates PCR products with blunt ends due to its 3´→5´ exonuclease activity. For direct cloning of the PCR products, the blunt-end cloning method should be used. When T vector cloning is required, the PCR products should be dA tailed by Taq DNA polymerase for 5-10 minutes at 72℃.n addition to fast amplification speed, high efficiency, high fidelity, and easy amplification of fragments up to 12 kb, EnzymoPure™ High-Fidelity DNA Polymerase can incorporate dUTP (2' -deoxy-uridine 5'-triphosphate) and dITP (2' -deoxy-inosine-5 '-triphosphate) bases into newly synthesized DNA. Please see Figure 1 and Figure 2 for its amplification effects.Figure 1. Amplification of target fragment by Aladdin's EnzymoPure™ High-Fidelity DNA Polymerase (EnzymoPure™) and EnzymoPure™ DNA Polymerase (EnzymoPure™) in PCR reactions containing dUTP or dITP. Reaction system of dUTP group (50µl)Application：Regular gene amplification with high fidelity; high fidelity NGS library preparation with high yield and low GC bias; amplification of bisulfite-converted DNA for bisulfite sequencing, deaminated DNA, or damaged DNA by formalin fixation and paraffin embedding for instance; preventing carryover contamination in PCR when combined with dUTP and uracil-N-Glycosylase (UNG) treatment. DNA stored for a long time will have random cytocine deamination, resulting in the inability of ordinary DNA polymerase to amplify, while this product can be compatible with and amplify the deaminated DNA[1-3].Source：Recombinant protein expressed in E. coli.Inactivation or inhibition：This enzyme can be removed and inactivated by phenol-chloroform extraction.Precautions：Because PCR reactions are extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommened for all PCR assays.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 Q6U™ High-Fidelity DNA Polymerase on ice.b. Set up the following reaction on ice. Mix well by gentle vortex or pipetting. Centrifuge briefly to collect liquid at the bottom of microcentrifuge tube.To amplify dsDNA 6kbReagentFinal concentrationVolumeFinal concentrationVolumeNuclease-free water-(38.5-x)µl-(32.5-y)µl10X Q6U™ Buffer1X5µl1X5µl dNTP (2.5mM each)0.25mM each5µl0.5mM each10µlTemplate DNA10pg-1µg*xµl10pg-1µg*yµlPrimer mix (10µM each)0.2µM each1µl0.4µM each2µlQ6U™ High-Fidelity DNA 1U/50µl0.5µl1U/50µl0.5µlTotal 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: 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. 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. c. (Optional) When using a thermal cycler without a heated lid, place a drop of mineral oil onto the top of PCR reaction mixture.2. Transfer PCR reactions to a thermal cycler 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 72ºC, but the amplification will be slightly reduced. For sequences difficult to amplify, denaturation at 92ºC and extension at 72ºC 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 extension time should be set according to the length of amplicons. For amplification of DNA fragments shorter than 6kb, the recommended extension time is 15 seconds per kb. For example, set 15 seconds to amplify amplicons of 1kb, 30 seconds for amplicons of 2kb, and so on. When amplifying DNA fragments longer than 6kb, the recommended extension time is 1 minute per kb (e.g. use 10 minutes to amplify amplicons of 10kb). 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.3. Analyze PCR products by agarose gel electrophoresisFAQ: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, methods such as touch down can be used to anneal, usually by gradually and slowly lowering the temperature from 65ºC to 55ºC or 50ºC to make the annealing more sufficient. 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 95ºC 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 can not.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 appropriatelyo. When non-specific fragments are produced, the annealing temperature can be increased appropriately.p. Positive and negative controls are always recommended when optimizing PCR reactions.2. Occurence of non-specific bands or DNA smear when examined by agarose gel electrophoresis.Increase the annealing temperature by 2-5℃.Reduce the amount of DNA template.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.Reduce the amount of Q6U™ High-Fidelity DNA Polymerase appropriately.Shorten the extension time appropriately... Read More | Inquire | Purity≥ 95% SDS-PAGE.Additional sequence informationMature chain.FunctionCould be a growth factor active in the process of wound healing. Acts as a mitogen in the lung. May act in a manner similar to FGF-7 | Purity>97% by SDS-PAGE and HPLC analyses.FunctionMay be involved in macrophage-mediated cellular proliferation. It is mitogenic for fibroblasts and smooth muscle but not endothelial cells. It is able to bind EGF receptors with higher affinity than EGF itself and is a far more potent mitogen for Purity>97% by SDS-PAGE and HPLC analyses.FunctionMay be involved in macrophage-mediated cellular proliferation. It is mitogenic for fibroblasts and smooth muscle but not endothelial cells. It is able to bind EGF receptors with higher affinity than EGF itself and is a far more potent mitogen for smooth muscle cells than EGF. Also acts as a diphtheria toxin receptor.Background:Human HB-EGF (Heparin-Binding EGF-like growth factor) is a 12-16 kDa member of the EGF family of peptide growth factors (1-3). Also known as the DTR (diphtheria toxin receptor), it is further classified as a group 2 ErbB ligand based on its ability to activate both the EGF/ErbB1 and ErbB4 receptors (4, 5). HB-EGF is synthesized as a 208 amino acid (aa) type I transmembrane preproprecursor (1, 6). It contains a 19 aa signal sequence, a 43 aa prosegment, an 86 aa mature region (aa 63-148), an 11 aa juxtamembrane cleavage peptide, a 24 aa transmembrane segment, and a 25 aa cytoplasmic tail (aa 184-208). As an integral membrane protein, HB-EGF is expressed as a 19-27 kDa protein in mammalian cells (7-9). The variability in molecular weight (MW) is attributed to heterogeneity in glycosylation and/or the utilization of multiple proteolytic cleavage sites during maturation. Mature HB-EGF is a soluble peptide that arises from proteolytic processing of the transmembrane form. It possesses an EGF-like domain between aa 104-144, and a heparin-binding motif between aa 93‑113. Although the aa range for "mature" HB-EGF is typically stated to be Asp63-Leu148, potential N-terminal start (cleavage) sites also exist at Gly32, Arg73, Val74, Ser77 and Ala82 (8, 10-12). Thus, differential processing (in part) likely accounts for the 16-23 kDa range in MW noted for mammalian-derived mature HB-EGF. Proteases suggested to contribute to HB-EGF processing include TACE, MMP-3 and -7, ADAM-17 and ADAM-12 (11, 13-16). When expressed recombinantly in E.coli, HB-EGF (aa 73-148) runs at 14 kDa in SDS-PAGE; when expressed in Baculovirus, HB-EGF (aa 63-148, 77-148 and 32-148) runs at 18 kDa, 15 kDa, and 19 kDa respectively (8, 12, 17). Over aa 63-148, human HB-EGF- shares 76% and 73% aa sequence identity with rat and mouse HB-EGF, respectively (1, 18). Cells known to express HB-EGF include bronchial epithelium (19), visceral and vascular smooth muscle (20, 21), CD4+ T cells (22), cardiac muscle (23), glomerular podocytes (24), keratinocytes (13) and IL-10-secreting regulatory macrophages (25). As noted earlier, HB-EGF is known to bind to both 170 kDa EGFR and 180 kDa ErbB4, and through heterodimerization, ErbB2 (13, 26). Activity associated with ErbB4 binding appears to be limited to non-mitogenic actions, while EGFR binding induces both mitogenic and non-mitogenic activity... Read More | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： Interleukin 33 (IL-33), also known as DVS27 or NF-HEV (Nuclear Factor from High Endothelial Venules), is a pro-inflammatory protein and a chromatin-associated cytokine of the IL-1 family with high sequencePurity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： Interleukin 33 (IL-33), also known as DVS27 or NF-HEV (Nuclear Factor from High Endothelial Venules), is a pro-inflammatory protein and a chromatin-associated cytokine of the IL-1 family with high sequence and structural similarity to IL-1 and IL-18. IL-33 protein is expressed highly and rather selectively by high endothelial venule endothelial cells (HEVECs) in human tonsils, Peyer's patches, and lymph nodes. IL-33 protein has transcriptional regulatory properties, and the researches suggested that IL-33 is a dual-function protein that might act both as a cytokine and as an intracellular nuclear factor. As a type 2 cytokines, IL-33 protein also play a pivotal role in helminthic infection and allergic disorders... Read More |