| 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 | 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula results in high yield, strong repeatability, and good stability of amplified products. This product does not contain dyes. After the PCR program is completed, an appropriate amount of sample buffer can be added as needed for electrophoresis operation. The amplified PCR product has an "A" base attached to the 3 'end, making it suitable for direct use in T/A cloning. Mainly suitable for PCR amplification of DNA, DNA sequencing and other experiments.Quality control: T665627Component5mlStorageT665627A2×Taq MasterMix5×1ml-20℃. Avoid freeze/thaw cycle.T665627BddH₂O5×1ml-20℃. Avoid freeze/thaw cycle.Notes: 2×Taq MasterMix contains Taq DNA Polymerase, 3mM MgCl2 and 400µM each dNTP After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes.Usage:The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.1. PCR reaction systemReagent50 µlReaction systemFinal concentration2×Taq MasterMix25 µl1×Forward Primer，10 µM2 µl0.4 µMReverse Primer，10 µM2 µl0.4 µMTemplate DNA<0.5 µg<0.5 µg/50 µlddH2Oup 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 conditionsStepTemperatureTime/Pre denaturation95℃2 min/Denaturation94℃30 s25-35 cyclesAnneal55-65℃30 s25-35 cyclesExtend72℃30 s25-35 cyclesFinally extended72℃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 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 | Inquire | Purity> 95 % by SDS-PAGE and HPLC analyses.FunctionPromotes cell proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up-regulating, in skin fibroblasts, the expression of a number of genes involved in angiogenesis, inflammation and matrix Purity> 95 % by SDS-PAGE and HPLC analyses.FunctionPromotes cell proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up-regulating, in skin fibroblasts, the expression of a number of genes involved in angiogenesis, inflammation and matrix remodeling including VEGA-A, VEGA-C, MMP1, MMP3, TIMP1, uPA, PAI-1 and integrins alpha-3 and alpha-5. CYR61-mediated gene regulation is dependent on heparin-binding. Down-regulates the expression of alpha-1 and alpha-2 subunits of collagen type-1. Promotes cell adhesion and adhesive signaling through integrin alpha-6/beta-1, cell migration through integrin alpha-v/beta-5 and cell proliferation through integrin alpha-v/beta-3.Banckground:Cyr61, also known as CCN1, is a 40-45 kDa matricellular glycoprotein that plays an important role in cellular adhesion and migration (1). Cyr61 consists of an IGFBP domain, a VWF type C domain, a TSP type I domain, and a cysteine knot domain (2). Mature human Cyr61 shares 93% amino acid sequence identity with mouse and rat Cyr61. It is widely expressed during development and in adult tissues (2, 3). Cyr61 associates with the extracellular matrix (ECM) and with many cell surface molecules including Integrins alpha V beta 3, alpha V beta 5, alpha M beta 2, and alpha 6 beta 1, Syndecan-4, and heparan sulfate proteoglycans (1, 3). Cyr61 mediates the adhesion and migration of multiple cell types and also promotes vascular endothelial cell tubule formation (4-6). Plasmin cleavage of ECM-bound Cyr61 releases a 28 kDa N-terminal fragment which retains the ability to promote endothelial cell migration (7). Cyr61 exhibits both tumorigenic and tumor suppressor properties. It is up-regulated and promotes tumorigenesis, angiogenesis, and metastasis in breast, renal, gastric, squamous cell, and colorectal carcinomas as well as in glioma (8-12). In contrast, whendown-regulated, it suppresses tumor growth in endometrial, hepatic, and non-small cell lung cancers (8, 13, 14). Cyr61 is also up-regulated in injured skin and bone where it induces the expression of growth factors, cytokines, proteases, and integrins involved in wound repair (15, 16)... Read More | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：HSPD1, also known as HSP60, is a member of the chaperonin family. HSPD1 may function as a signaling molecule in the innate immune system. This protein is essential for the folding and assembly of newly Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：HSPD1, also known as HSP60, is a member of the chaperonin family. HSPD1 may function as a signaling molecule in the innate immune system. This protein is essential for the folding and assembly of newly imported proteins in the mitochondria. It may also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix. HSPD1 gene is adjacent to a related family member and the region between the 2 genes functions as a bidirectional promoter. Several pseudogenes have been associated with this gene. Mutations associated with this gene cause autosomal recessive spastic paraplegia 13. Defects in HSPD1 are a cause of spastic paraplegia autosomal dominant type 13 (SPG13). Spastic paraplegia is a degenerative spinal cord disorder characterized by a slow, gradual, progressive weakness and spasticity of the lower limbs. Defects in HSPD1 are the cause of leukodystrophy hypomyelinating type 4 (HLD4); also called mitochondrial HSP60 chaperonopathy or MitCHAP-60 disease. HLD4 is a severe autosomal recessive hypomyelinating leukodystrophy. HSPD1 is clinically characterized by infantile-onset rotary nystagmus, progressive spastic paraplegia, neurologic regression, motor impairment, profound mental retardation. Death usually occurs within the first two decades of life... Read More |