| Description | The 6X His Peptide is commonly used for the competitive elution of His tag fusion proteins bound to His antibodies. It can be used to elute His tag fusion proteins from Anti-His Magnetic Beads and conventional Anti-His antibodies during immunoprecipitation.This product competitively elutes the 6X The 6X His Peptide is commonly used for the competitive elution of His tag fusion proteins bound to His antibodies. It can be used to elute His tag fusion proteins from Anti-His Magnetic Beads and conventional Anti-His antibodies during immunoprecipitation.This product competitively elutes the 6X His fusion protein. The eluted protein sample does not contain the light and heavy chains of Anti-His antibody, thus effectively eliminating the interference of anibodies with downstream Western blot analysis.The amino acid sequence of 6X His Peptide is His-His-His-His-His-His (HHHHHH).The main parameters of 6X His Peptide are as followsPrecautions：The solution of this product should be aliquoted and stored at -20℃ or lower temperatures to avoid repeated freeze-thaw cycles.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. For 25mg provided in powder form, centrifuge at 8,000-12,000×g for 10-30 seconds before opening to collect the powder at the bottom of the tube. Dissolve it in TBS by gently pipetting or shaking. Do not vortex vigorously to avoid denaturation and inactivation of the peptide.2. P9811-1mg and P9811-5mg can be directly used for preparing elution buffer. 3. Competitive elution with the 6X His Peptide: This elution method is non-denaturing, with high elution efficiency, and the eluted sample does not contain the light and heavy chains of His antibodies.a. Preparation of 6X His tag elution buffer: Based on the required amount of elution buffer, dilute an appropriate amount of 6X His Peptide stock solution with TBS to a final concentration of 150µg/ml.b. Add 100µl of 6X His tag elution buffer (150µg/ml) to each immunoprecipitated sample, and incubate on ice or at 4℃ for 30 minutes to 2 hours with shaking. In order to improve the elution efficiency, the incubation time can be extended or the elution can be repeated. The volume of the 6X His tag elution buffer is generally 5 times that of the bead or gel suspension.c. Centrifuge at 6000×g for 30 seconds at 4℃, and carefully transfer the supernatant to a new tube. The supernatant contains the 6X His fusion protein and its protein complexes. Be careful not to touch the resin when taking the supernatant. 4. Store the eluted 6X His fusion protein at 4℃ for immediate use, or at -20℃ for long-term storage... Read More | Product contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a preProduct contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 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 systemreagents50µl reaction systemfinal concentration2×Fast Probe Mixture25 µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10 µM1µl0.2µM²⁾Template DNA2µl³⁾ 50x Low ROX or High ROX(optional)⁴⁾1µl1×ddH₂Oup to 50µlNote: 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 to allow the starting template to fully unchain.(2) It is recommended to use two-step PCR reaction program, if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification, and the annealing temperature, please use the range of 56 ℃ - 64 ℃ as a setting reference... Read More | Inquire | Inquire | Inquire |