| 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 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.sApplication：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)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... Read More | Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high amplification efficiency and low mismatch rate, and can efficiently amplify DNA fragments. Most of the PCR products amplified with this product contain an "A" base at the 3 'end, which can be directly used for T/A cloning. This product is suitable for conventional PCR reactions and gene cloning reactions that require high fidelity. E665597Component500 UStorageE665597AEs Taq DNA Polymerase, 5 U/µL 100 µL -20℃. Avoid freeze/thaw cycle.E665597B10×PCR Buffer 1.8 mL -20℃. Avoid freeze/thaw cycle.Activity definition:Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.Quality control:After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one month without significant changes in activity.1. PCR reaction system Reagent 50 µlReaction system Final concentration 10×PCR Buffer 5 µL 1× dNTP Mix，10 mM each 1 µL 200 µM each Forward Primer，10 µM 2 µL 0.4 µM Reverse Primer，10 µM 2 µl 0.4 µM Template DNA <0.5 µg <0.5 µg/50 µl Es Taq DNA Polymerase，5 U/µl 0.25-0.5 µl 1.25-2.5U/50 µl ddH2O up to 50 µL /Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions Step Temperature Time / Pre denaturation 94℃ 2 min / Denaturation 94℃ 30 s 25-35 cycles Anneal 55-65℃ 30 s 25-35 cycles Extend 72℃ 30 s 25-35 cycles Finally extended 72℃ 2 min / Attention:1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of Es Taq DNA Polymerase in this product is 2 kb/min.3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible... Read More | Product contentF665766Component5 mLStorageF665766A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665766BddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Product contentF665766Component5 mLStorageF665766A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665766BddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemReagents50 µl Reaction systemFinal concentration2×Fast Probe Mixture25 µl1×Forward Primer，10 µM1 µl0.2 µM¹⁾Reverse Primer，10 µM1 µl0.2 µM¹⁾Probe，10 µM1 µl0.2 µM²⁾Template DNA2 µl³⁾ 50×Low ROX or High ROX（optional）⁴⁾1 µl1×ddH₂Oup to 50 µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes in order to make the starting template fully unchained.(2) It is recommended to use two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out a three-step PCR amplification, annealing temperature, please use the range of 56 ℃ - 64 ℃ for as a reference for the setting... Read More | Extinction Coeff.A280 nm = 1.0 at 1.0 mg/mLSpecificityMonospecific for Factor B in human plasma and serumGeneral DescriptionProduct is whole polyclonal antiserum from goats immunized with highly purified human complement protein. Product is not a purified IgG fraction. Goats are maintained in FDA Extinction Coeff.A280 nm = 1.0 at 1.0 mg/mLSpecificityMonospecific for Factor B in human plasma and serumGeneral DescriptionProduct is whole polyclonal antiserum from goats immunized with highly purified human complement protein. Product is not a purified IgG fraction. Goats are maintained in FDA certified facilities.Physical Characteristics & StructureAntibodies present in the antisera are primarily IgGApplicationsWestern Blots: Effective at dilutions 1/4,000 to 1/8,000 depending on conditions.Most effective against non-reduced antigen.ELISA: Effective at dilutions 1/8,000 to 1/16,000 depending on conditions.Immunodiffusion: Effective against NHS and plasma at 1/16 dilution... Read More | Purity: >90%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description: Major histocompatibility complex, class II, DR alpha (HLA-DRA) belongs to the MHC class II family. HLA-DRA binds peptides derived from antigens which access the endocytic route of antigen presenting cells (APC) Purity: >90%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description: Major histocompatibility complex, class II, DR alpha (HLA-DRA) belongs to the MHC class II family. HLA-DRA binds peptides derived from antigens which access the endocytic route of antigen presenting cells (APC) and presents them on the cell surface for identification by the CD4 T-cells. The peptide binding cleft accommodates peptides of 10-30 residues. The peptides presented by MHC class II molecules are generated mainly by degradation of proteins which access the endocytic route, where they are processed by lysosomal proteases and other hydrolases... Read More |