| 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 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.sApplication：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... Read More | Inquire | Inquire | Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw Products contentN665730Component24 T96 TStorageN665730ATPS V50 144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730B5×FA Reaction Buffer144 µL576 µL-20℃. Avoid freeze/thaw cycle.N665730C2×HiFidelity PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle.N665730DPPM48 µL192 µL-20℃. Avoid freeze/thaw cycle.* This kit is suitable for human genomic DNA library construction with a starting template DNA input of 50 ng. We also have transposase library construction kits for human genomic DNA starting at 5 ng and 1 ng, so it is recommended to use different kits for different starting amounts of DNA in order to obtain higher quality libraries. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for DNA libraries with a starting template of 50 ng, and all reagents in the kit have been subjected to strict quality control and functional validation to maximize the stability and reproducibility of library construction. Product Features ● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification-preferred steps.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kits: transposase method for second-generation sequencing multi-sample primer kits are recommended. 4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.Sample PreparationDNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).Schematic diagram of DNA banking processprocedureDNA fragmentation, junction reaction1. Add the following reagents to a 200 µl PCR tube: 2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:DNA should be purified immediately after the fragmentation reaction has been performed and the transposase is still in a high state of activity.to prevent smaller library fragments due to DNA over-fragmentation. Purification of fragmentation productsWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Add 50 µl of magnetic beads equilibrated to room temperature to the fragmentation product, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, then add 23 µlddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer 21 µl of supernatant to a new 200 µl PCR tube.PCR amplification Add the following reagents to the 200 µl PCR tube: Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads to be used is different, please refer to the attached table for the specific amount of magnetic beads to be used (if other brands of magnetic beads are used, you need to find out the optimal amount of magnetic beads to be used on your own).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, the amplification products can also be purified without selective recovery of DNA fragments as described on page 6 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR product to a 1.5 ml centrifuge tube, rehydrate to 100 µl and add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the centrifuge tube from the magnetic rack, vortex and oscillate to completely resuspend the beads, and let stand at room temperature for 5 minutes. Leave brieflycentrifuge, place the tube on a magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recoveryLibrary DNA fragment purificationWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library. Average total length of librariesApproximate conversion formula Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More | Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. It is derived from a 34 kDa inactive precursor zymogen, trypsinogen, after enzymatic removal of an N-terminal 6-amino acid leader sequence resulting in the 23.8 kDa Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. It is derived from a 34 kDa inactive precursor zymogen, trypsinogen, after enzymatic removal of an N-terminal 6-amino acid leader sequence resulting in the 23.8 kDa trypsin molecule. The optimum pH is 8.0. Trypsin is inhibited by organophosphorus compounds such as diisopropylfluorophosphate and natural inhibitors from pancreas. Soybean, lima bean, and egg white are also sources of natural inhibitors. Trypsin cleaves amide and ester bonds of Arg and Lys. The Aladdin Sequencing Grade Trypsin has been further purified to remove trace contaminating proteases and autolysis products which could interfere in trypsin digestion experiments, and exhibits a single band on PAGE.Trypsin is a serine protease used to hydrolyze proteins. Trypsin from bovine pancreas has a molecular weight of 23.8 kDa. Trypsins are used for the re-suspension of cells during cell culture and in proteomics research for the digestion of various proteins... Read More |