| Product 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 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 ＜ 6kbTo 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 ＜6kbTo 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.
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