| Product Description | aladdin UltraBio™ Blood Direct PCR Master Mix (HF, 2X) is designed specially to perform PCR directly from whole blood samples without DNA extraction. It is a 2X concentrated solution of EnzymoPure™ HF 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 reactions to be directly loaded on an agarose gel for electrophoresis.Whole blood can be added directly into PCR reactions with no prior DNA extraction or sample preparation. aladdin UltraBio™ Blood Direct PCR Master Mix (HF, 2X) employs the EnzymoPure™ HF DNA polymerase that exhibits extremely high tolerance of various inhibitors present in human or mouse blood. Fresh blood, blood stored at 4ºC or frozen, and preserved with EDTA, citrate or heparin are all suitable for 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, use 1μl of whole blood generally. For the detection of bacterial, viral and other microbial infections, up to 4μl of whole blood can be used to improve the detection sensitivity. For some special type of whole blood, such as human blood with heparin anticoagulant, 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. To obtain the best detection result, we recommend using the EDTA anticoagulant, 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 (HF, 2X) can tolerate 50%, 20% and 40% blood preserved with heparin, citrate and EDTA, respectively. This product can also tolerate more than 20% of mice blood. The blood concentration in PCR reactions can be 1-20%, and 5-10% is regularly used.Figure 1. Agarose gel electrophoresis of amplicons from different percentages of human blood preserved with heparin, citrate or EDTA anticoagulants, using Aladdin's UltraBio™ Blood Direct PCR Master Mix (HF, 2X) . Control primers provided in this product are used to amplify the 237bp DNA fragment upstream of the non-coding region of SOX21. 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.Similar to EnzymoPure™ DNA Polymerase , the EnzymoPure™ HF DNA Polymerase (Blood-resistant) contained in this product has a higher fidelity than Pfu DNA Polymerase . Therefore, this product can be used for both general PCR detection and gene cloning.DNA fragments of 5kb or longer can be amplified by this product from citrate blood. The DNA amplicons in different lengths are shown in Figure 2.aladdin UltraBio™ Blood Direct PCR Master Mix (HF, 2X) generates blunt-end PCR products which must be dA-tailed with Taq DNA polymerase before TA cloning.s
Application： DNA amplification from whole blood; genotyping; detection of bacterial, viral and other microbial infection in blood samples; genotype analysis of transgenic mice. Compared to HemoTaq™ DNA Polymerase , HemoTaq™ HF DNA Polymerase contained in this product is a high-fidelity enzyme and is more suitable for gene cloning.Inactivation or inhibition: Activities of HemoTaq™ HF DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction.
Inactivation or inhibition： Activities of HemoTaq™ HF 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.Preserved blood with anticoagulants can be added directly into the PCR reaction without extra preparation. The recommended blood concentration is approximately 5%.After amplification, centrifuge the PCR reaction at 3000-5000×g for 3-5 minutes to collect the supernatant for agarose 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. Please use the Pure™ Ultrapure Water (, BST876) 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. Prepare PCR reactions:a. Thaw the Easy-Load™ Blood Direct PCR Master Mix (HF, 2X) and Control Primer mix (10μM each) 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 (HF, 2X)1025μl1XPCR Primer Mix (10µM each)12.50.5μM eachWhole Blood with Anticoagulantsxy-Total Volume2050-Note 1: Blood concentration in PCR reaction mixture: 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 the reaction to amplify DNA with high GC content. For dried blood spot samples, 0.8 mm2 and 2 mm2 are recommended for 20μl and 50μl PCR reaction volumes, 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. a.Mix well by gentle vortex or pipetting. Centrifuge briefly to allow liquid to accumulate at the bottom of the PCR tube.b. (Optional) When using a thermocycler without a hot lid, add a drop of mineral oil to the reaction to avoid evaporation. 2. Transfer PCR reactions to a thermal cycler and run thermocycling conditions as follows:StepTemperatureAmplicon (＜1kb)Amplicon (＞1kb)Step1( Initial Denaturation)94℃5min5minStep2(Denaturation)94℃30sec30secStep3(Annealing)55℃30sec30secStep4(Extension)68℃15sec/kb1min/kbStep5 (Go to Step2, cycles)Go To Step 2, 30-40 cyclesGo To Step 2, 30-40 cyclesStep6(Final Extension)68℃10min15minStep7(Holding)5℃foreverforeverNote 1: Denaturation at 94ºC 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 obtaining of the expected DNA fragments. Use 30-35 cycles for gene amplification, or 35-40 cycles for detection of microbial contamination.3. Analyze PCR products by agarose gel electrophoresis: After amplification, centrifuge the PCR reaction at 3000-5000×g for 3-5 minutes to collect the supernatant for agarose gel electrophoresis. This product contains premixed loading dye that enables loading PCR reactions directly onto 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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