| Product Description | Aladdin's UltraBio™ Multiplex PCR Master Mix (2X) is designed specially for simultaneous, well-balanced amplification of multiple DNA targets in a single PCR reaction with high efficiency, specificity and sensitivity. It is a 2X concentrated solution of Mult DNA polymerase, dNTPs, and all of the components required for PCR, except DNA template and primers. The premix also contains a 2X loading buffer that enables PCR reactions to be directly loaded on an agarose gel for electrophoresis. This product can be used not only for ordinary multiplex PCR, but also for direct PCR of whole blood, serum or plant samples.ItemComponentQuantityD7305S-1UltraBio™ Multiplex PCR Master Mix (2X)1mlD7305S-2Control Template and Primer Mix25μlManual-1 copyMultiplex PCR technique has been widely used in scientific research, disease diagnosis and diagnostic genotyping, such as quantitative or semi-quantitative gene expression analysis using cDNA as template, multi-gene detection of trace samples of animal, plant, fungi, bacteria or viruses.ItemComponentQuantityD7305M-1UltraBio™ Multiplex PCR Master Mix (2X)1ml×5D7305M-2Control Template and Primer Mix50μlManual-1 copyThis product has superior multiplex amplification performance. This product can easily achieve the simultaneous, well-balanced amplification of 15 target DNA fragments in a single PCR reaction with high efficiency, specificity and sensitivity (Figure 1). Template as low as 1pg can be amplified successfully with only 30 PCR cycles (Figure 1). Regular PCR reagents normally have a bias towards different primers and DNA templates, while the Mult DNA Polymerase has been diligently optimized for multiplex PCR to ensure well-balanced amplification of multiple target DNA fragments with high efficiency, specificity and sensitivity.This product can simultaneously amplify 15 different DNA fragments from sample templates as low as 100pg (Figure 1).Figure 1. Agarose gel electrophoresis of multiplex PCR products from different amounts of Lamda DNA using Aladdin's UltraBio™ Multiplex PCR Master Mix (2X) . Fifteen primer pairs with a final concentration of each primer at 0.2μM are used to amplify DNA fragments of 113bp, 143bp, 199bp, 253bp, 303bp, 406bp, 501bp, 576bp, 710bp, 802bp, 903bp, 989bp, 1167bp, 1300bp and 1463bp, respectively. M, DNA marker . As shown in the figure, 15 different DNA fragments can be amplified from Lamda DNA as low as 10pg.This product is compatible with blood and plant samples. Whole blood, serum or plant samples can be added directly to PCR reactions without DNA extraction and purification. Both dried blood spots on cards and blood with EDTA, heparin or sodium citrate anticoagulants are compatible with this product. Positive control template and primer mix are provided for troubleshooting. The Control Template and Primer Premix contains 15 primer pairs used to amplify DNA fragments with different size ranging from 113bp to 1463bp.This product generates PCR products with 3'-dA overhangs, which can be used for TA cloning subsequently. The Mult DNA polymerase contained in the Master Mix has a fidelity similar to Taq DNA polymerase and is therefore recommended primarily for qualitative and semi-quantitative analysis.s
Application： DNA amplification; multiplex PCR detection of bacterial or viral infection and gene mutation in blood and plant samples.Storage Conditions：Store reagents at -20℃ upon receipt. Avoid repeated freeze-thaws.Precautions：Primer design is the key to a successful multiplex PCR. Primers need to meet the conventional primer rules to avoid non-specific amplification and non-amplification, and should be verified by PCR individually. The melting temperature of all primers for multiplex PCR should be more than 60ºC. Better multiplex PCR results are normally obtained using primers with Tm higher than 65℃. The Tm value is calculated according to the formula: Tm (℃) = 2n(A) + 2n(T) + 4n(G) + 4n(C) (for example, for a primer contains 3 A, 7 T, 4 G and 6 C, Tm = 2×3 + 2×7 + 4×4 + 4×6 = 60℃).Primers should be desalted, PAGE or HPLC-purified. It is recommended to prepare a primer mix solution including forward and reverse primers at 10μM each.An extension time of two minutes per kb is recommended. For DNA targets hard to be amplified, try 3-4 min per kb.For abundant PCR products, it is recommended to load 2μl of PCR reaction for agarose gel electrophoresis analysis. Too many PCR products may lead to uneven DNA bands.Because the multiplex PCR reaction is extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.We recommend using ’s Multiplex PCR Kit and PCR Enhancer to amplify GC-rich DNA fragments.Nuclease-free water is not provided in this product. Use the Ultrapure Wateror other types of ultra-pure H2O.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.
Precautions： Primer design is the key to a successful multiplex PCR. Primers need to meet the conventional primer rules to avoid non-specific amplification and non-amplification, and should be verified by PCR individually. The melting temperature of all primers for multiplex PCR should be more than 60ºC. Better multiplex PCR results are normally obtained using primers with Tm higher than 65℃. The Tm value is calculated according to the formula: Tm (℃) = 2n(A) + 2n(T) + 4n(G) + 4n(C) (for example, for a primer contains 3 A, 7 T, 4 G and 6 C, Tm = 2×3 + 2×7 + 4×4 + 4×6 = 60℃).Primers should be desalted, PAGE or HPLC-purified. It is recommended to prepare a primer mix solution including forward and reverse primers at 10μM each.An extension time of two minutes per kb is recommended. For DNA targets hard to be amplified, try 3-4 min per kb.For abundant PCR products, it is recommended to load 2μl of PCR reaction for agarose gel electrophoresis analysis. Too many PCR products may lead to uneven DNA bands.Because the multiplex PCR reaction is extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.We recommend using ’s Multi™ Multiplex PCR Kit and Multi™ PCR Enhancer to amplify GC-rich DNA fragments.Nuclease-free water is not provided in this product. Use the Pure™ Ultrapure Water or other types of ultra-pure H2O.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. Primer design:Good primers are crucial for a successful multiplex PCR. We recommend using primer design tools for primer design. Primers should comply with the following rules:a. Primers are generally 20-30 nucleotides in length; b. Primers have a GC content of 40-60% (preferably 50-60%);c. Try to avoid complementary sequences at the 3’-end of all primers, runs of three or more G/C at the 3' end, and secondary structures within primers;d. The melting temperature of all primers for multiplex PCR should be more than 60°C according to the formula: Tm (°C) = 2n(A) + 2n(T) + 4n(G) + 4n(C). Primers with Tm between 65-68°C are preferred and the difference in Tm of primer pairs should be within 5-6℃.e. It is recommended that the target fragment should not exceed 1500bp. Although the target fragment of approximately 1500bp can be well amplified, it is more likely to produce uneven bands when long fragments and short fragments are amplified simultaneously.f. Primers should be desalted, PAGE or HPLC-purified, as the primer quality is a critical factor for good multiplex PCR. Primers should be dissolved in TE buffer (10 mM Tris-HCl, 1.0 mM EDTA, pH 8.0).2. Preparation of primer stock:Prepare primer stock at 100µM in TE buffer for each primer, and then mix each primer pair at a 1:1 ratio in an appropriate amount of ultra-pure H2O to produce a primer pair of 10µM each.3. Preparation of PCR reactions:a. Thaw the Easy-Load™ Multiplex PCR Master Mix (2X) and Control Template and Primer Mix at room temperature. Mix well gently by inversion and centrifuge briefly. Keep the reagents on ice.b. Assemble PCR reactions on ice as follows:ComponentSampleControlSampleControlFinal concentrationNuclease-Free Water(10-x-0.4n)μl5μl(25-x-n)μl12.5μl-Easy-Load™ Multiplex PCR Master Mix (2X)10μl10μl25μl25μl1XPrimer Mix (10μM each)0.4μl×n-1μl×n-0.2μM eachTemplatexμl-xμl--Control Template and Primer Mix-5μl-12.5μl-Total volume20μl20μl50μl50μl-Note: (a) ‘n’ represents the number of primer pairs, that is the number of DNA targets. (b) The amount of DNA template has a significant influence on PCR amplification. For DNA samples with high complexity, such as mammalian genome DNA, 5ng-0.5μg of DNA template is recommended in 20μl of PCR reaction. For low complexity DNA, such as Lamda DNA or plasmid DNA, 5pg-5ng of DNA template is recommended in 20μl of PCR reaction. (c) For blood samples: The recommended blood concentration is 1-20%, and 5% can be used as a starting point. For detecting genomic DNA fragments in blood, lower blood concentration can be used. To detect the viral, bacterial or other microbial DNA in blood samples, a higher blood concentration is recommended. We recommend using Multiplex PCR Kit and PCR Enhancer for the amplification of DNA with high GC content, or adding 1−10% DMSO in PCR reactions. For dried blood spot samples, 0.8 mm2 and 2 mm2 are recommended for 20μl and 50μl of PCR reactions, respectively. (d) For plant samples: When setting up PCR reactions, 0.1-1mm and 0.3-3mm diameter leaves or other tender plant tissues of similar size are recommended for 20μl and 50μl of PCR reactions, respectively. Fresh and tender seeds are suitable for this Master Mix, but not dry and hard seeds. Use a clean scalpel to remove the seed shell, cut out 0.5-2mm diameter tissue, and put it directly into the PCR reaction. One to two small seeds such as tomato seeds, can be added directly into PCR reactions for DNA amplification. In 50μl of PCR reactions, the diameter of plant leaf or seed should not exceed 3mm, as too many PCR inhibitors introduced by plant samples will inhibit PCR amplification. It is recommended to perform two parallel PCR reactions for the same sample to reduce variations of sampling. To ensure the uniformity of sampling, it is recommended to use a specialized punch or scalpel for sampling, and to prevent cross-contamination by cleaning the punch or scalpel with 2% sodium hypochlorite solution between each time of sampling. To amplify DNA with high GC content, DMSO can be added to PCR reactions at a final concentration of 1-10% (v/v). (e) Each primer at a final concentration of 0.5 μM normally works well, but primer concentration can be optimized between 0.05-0.4 μM. Increase the primer concentration when amplification efficiency is low and decrease the primer concentration when non-specific PCR products are amplified. c. Mix the reaction by gentle vortex or pipetting. centrifuge briefly to allow liquid to accumulate at the bottom of the PCR tube.d. (Optional) When using a thermocycler without a hot lid, add a drop of mineral oil to the reaction to avoid evaporation.e. Transfer the PCR reactions to a thermal cycler and run thermocycling conditions as follows:StepTemperatureDurationCyclesInitial Denaturation94℃5 min1Denaturation94℃30 sec30-40Annealing60℃30 secExtension68℃2 min/kbFinal Extension68℃10 min1Holding4℃--Note 1: Optimize PCR running conditions based on the template, primer sequence, amplicon length and GC content, etc. The annealing temperature can be adjusted between 55-60℃. Touch down method can also be applied to make annealing more efficient. Note 2: Use 35 cycles for the first time to ensure the expected DNA fragments are amplified. The cycle number can be adjusted appropriately based on multiplex PCR results. 4. Analyze PCR products by agarose gel electrophoresis: The Master Mix contains premixed loading dye that enables reactions to be loaded directly on agarose gel for electrophoresis. When necessary, centrifuge PCR reactions at 3000-5000×g for 3-5 minutes to collect the supernatant for gel electrophoresis. Refer to Figure 1 for the multiplex PCR results of positive control.FAQ:1. No product at all or low yield.a. Primer sequence is not well designed. Use primer design tools 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 well but not your primers, redesign primers.b. DNA may have a high GC content. High GC genes are relatively difficult to be amplified. We recommend using Multiplex PCR Kit and PCR Enhancer for the amplification of GC-rich DNA fragments.c. Some DNA fragments are too long to be amplified. Although Multiplex DNA polymerase can amplify DNA fragments up to 5 kb in length, it is more suitable for the amplification of DNA fragments less than 1.5 kb in multiplex PCR reactions. Multiplex PCR does not work very well for the amplification of long DNA fragments. d. The presence of secondary structure in primers, primer dimmers or short primers, may result in poor annealing of primers to the target sequence. Try touch-down or other methods for annealing. A gradual cooling from 65ºC to 55ºC or 50ºC can usually make annealing more efficient.e. Annealing temperature needs to be optimized. If necessary, use a temperature gradient to find the optimal annealing temperature. f. Insufficient extension time. Use extension time of two minutes per 1000 base pairs. For DNA fragments hard to be amplified, try 3-4 min per kb. g. Possible problems of PCR thermal cycler. Use a different thermal cycler. h. PCR cycle number is insufficient. Try more PCR cycles. Not to exceed 40 cycles in general.i. The amount of DNA template is too low. Add more DNA templates but within the tolerance limit of the Master Mix.j.Use desalted, PAGE or HPLC-purified PCR primers. k. Use dNTP mix in high quality.l. Add more DNA polymerase appropriately.m. When non-specific DNA fragments are amplified, increase the annealing temperature appropriately.n. Use Positive Control Template and Primers provided to ensure that PCR conditions are optimal.o. Make sure all components necessary for multiplex PCR are added. 2. Presence of non-specific PCR products or DNA smear.a. Increase the annealing temperature by 2-5ºC.b. Adjust PCR cycles.c. Reduce the amount of DNA template.d. Assemble the PCR reactions on ice. Non-specific products are produced easily if PCR reactions are set up at room temperature.e. Use less amount of DNA polymerase, or use Multiplex PCR Kit and PCR Enhancer .f. Decrease the extension time appropriately.
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