| Product Description | Aladdin's UltraBio™ Blood Direct PCR Master Mix (2X) is designed specially to perform PCR directly from whole blood samples. It is a 2X concentrated solution of Taq DNA Polymerase, dNTPs, and all of the components required for PCR, except DNA template and primers. The mix also contains a 2X loading buffer that enables PCR reactions to be directly loaded on an agarose gel for electrophoresis.Whole blood can be used directly as DNA template with no prior DNA extraction or sample preparation. aladdin UltraBio™ Blood Direct PCR Master Mix (2X) employs the heat-resistant EnzymoPure™ DNA polymerase that exhibits extremely high resistance to various inhibitors present in human or mouse blood. Fresh blood, blood stored at 4℃ or frozen, and preserved with EDTA, citrate or heparin are all compatible with this product, as is blood dried onto commercially available cards.This product is compatible with a wide range of blood concentrations ranging from 5% to 20%, and in some cases even up to 50%. In a 20μl of PCR reaction volume, add 1μl anticoagulant whole blood generally. For the detection of bacterial, viral and other microbial infections, up to 4μl whole blood can be added to improve the detection sensitivity. For some special types of whole blood, such as human blood with heparin, up to 10μl can be used, which is 50% of the PCR reaction volume.This product is compatible with three common anticoagulants including EDTA, sodium citrate, and heparin. If the best detection results are required, EDTA anticoagulant is recommended, as EDTA can inhibit nuclease activity by chelating metal ions. The PCR results of blood samples with three anticoagulants are shown in Figure 1. The UltraBio™ Blood Direct PCR Master Mix can tolerate 50%, 20% and 30% blood preserved with heparin, citrate and EDTA, respectively. This product can also tolerate more than 20% of mice blood. The recommended blood concentration in PCR reactions is 1-20%, and 5-10% is regularly used.Figure 1. Agarose gel electrophoresis of PCR products amplified by Aladdin's UltraBio™ Blood Direct PCR Master Mix (2X) using human blood containing different percentages of heparin, citrate or EDTA as template. Control primers provided in this product were used to amplify the 300bp DNA fragment in b-actin gene. M, DNA ladder .This product provides a pair of universal positive control primers that are compatible with many mammalian and other vertebrate species, by which the 237bp DNA fragment upstream of the non-coding region of SOX21 is amplified.The fidelity of EnzymoPure™ DNA Polymerase (Blood-resistant) contained in this product is similar to that of Taq DNA polymerase. To clone genes, we recommend using Aladdin's EnzymoPure™ HF DNA Polymerase or UltraBio™ Blood Direct PCR Master Mix (HF, 2X) .DNA fragment up to 5kb can be amplified by this product from preserved citrate blood. The DNA amplicons in different sizes are shown in Figure 2.Figure 2. Agarose gel electrophoresis of PCR products amplified by Aladdin's UltraBio™ Blood Direct PCR Master Mix (2X) using whole blood with 10% citrate as the template. M, DNA ladder ; 1, b-actin (300bp); 2, CCR5 (630bp); 3, CCR5 (1100bp); 4, CCR5 (2000bp); 5, IL-6 (2841bp); 6, IL-6 (4171bp); 7, IL-6 (4882bp).This product generates PCR products with 3'-dA overhangs, which can be used for TA cloning subsequently.s
Application： DNA amplification from whole blood; genotyping; detection of bacterial, viral and other microbial infection in blood samples; genotype analysis of transgenic mice.Inactivation or inhibition: Activities of HemoTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction.
Inactivation or inhibition： Activities of HemoTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction.
Precautions： Because the PCR reaction is extremely sensitive, contamination must be avoided during preparation of the PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.Anticoagulant blood can be added directly into PCR reactions without additional preparation. The recommended blood concentration is 5-10%.After amplification, centrifuge the PCR reaction at 3000-5000×g for 3-5 minutes and take the supernatant for gel electrophoresis. It is normal to have transparent gelatinous precipitations at the bottom of the PCR reactions because of the presence of anticoagulants.Nuclease-Free water is required but not supplied. Use any type of ultra-pure H2O or purchase the Pure™ Ultrapure Water (, ST876).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 the Easy-Load™ Blood Direct PCR Master Mix (2X) and Control Primer mix (10μM) at room temperature. Mix well gently by inversion and centrifuge briefly. Keep the reagents on ice.b. Assemble PCR reactions on ice as follows:ComponentVolume (μl)Volume (μl)Final Nuclease-Free Water9-x 22.5-y-Easy-Load™ Blood Direct PCR Master Mix (2X)1025μl1XPCR Primer Mix (10µM each)12.50.5μM eachWhole Blood with Anticoagulantsxy-Total Volume2050-Note 1: Blood concentration in PCR reactions: 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 adding 1−10% DMSO to PCR reactions to amplify DNA with high GC content. For dried blood spot samples, use 0.8 mm2 and 2 mm2 in 20μl and 50μl PCR reaction volume, respectively.Note 2: Each Primer at a final concentration of 0.5 μM normally works well, but primer concentration can be optimized between 0.1-1.0 μM. Increase the primer concentration when amplification efficiency is low and decrease the primer concentration when non-specific PCR products are amplified. c. Mix well 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. 2. Transfer the PCR reactions to a thermal cycler and run thermocycling conditions as follows: StepTemperatureDurationCyclesInitial Denaturation94℃5 min1Denaturation94℃30 sec30-40Annealing55℃30 secExtension68℃2 min/kbFinal Extension68℃10 min1Holding4℃--Note 1: Denaturation at 94℃ for 5 min is to lysis the blood cells to release genomic DNA.Note 2: Optimize PCR running conditions based on the template, primer sequence, amplicon length or GC content, etc. Note 3: Use 35 cycles for your first try to ensure the amplification of the expected DNA fragment.3. Analyze PCR products by gel electrophoresis: After amplification, centrifuge the PCR reactions at 3000-5000×g for 3-5 minutes and take the supernatant for agarose gel electrophoresis. This product contains premixed loading dye that enables reactions to be loaded directly on the agarose gel.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. In this case, GC-Rich buffer suitable for amplifying GC-rich DNA fragments is recommended, and PCR reaction parameters should be adjusted accordingly.c. DNA fragment is too long to be amplified. Although HemoTaq™ DNA polymerase contained in the Master Mix can amplify DNA fragments up to 5 kb in length, it is more suitable for amplification of DNA fragments less than 2-3 kb. Optimize primer sequence or other PCR parameters to amplify longer 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 efficiently.e. Annealing temperature needs to be optimized. If necessary, use a temperature gradient to find the optimal annealing temperature for each template-primer pair combination. f. Insufficient extension time. Use an 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 blood but within the tolerant limit of the Master Mix, or try nested PCR or secondary PCR. Nested PCR is to design a pair of PCR primers inside the originally designed PCR primers, and then conduct PCR amplification again with the diluted first PCR product as a template. Instead, secondary PCR uses the same primers for PCR amplification again with the diluted first PCR product as a template. Nested PCR can usually remove the non-specific DNA amplification, but secondary PCR can not. j. Use desalted, PAGE or HPLC-grade PCR primers. k. When non-specific DNA fragments are amplified, increase the annealing temperature appropriately.l. Positive control and negative control are always recommended when optimizing PCR reactions. 2. Presence of non-specific PCR products or DNA smear.a. Increase the annealing temperature by 2-5ºC.b. Reduce the blood concentration.c. Assemble the PCR reactions on ice. Non-specific products are produced easily if PCR reactions are set up at room temperature.d. Supplement the PCR reactions with an appropriate amount of DMSO or other reagents that can improve the amplification of GC-rich templates.e. Decrease the extension time appropriately.
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