A shorter probe increases quenching efficiency by bringing the fluorophore and quencher closer together, resulting in lower background and higher signal for greater sensitivity and precision. With the added stabilization, the probe itself can be designed significantly shorter. MGB stabilizes van dar Waals forces, which increases the binding affinity between the probe and target and increases the melting temperature (T m). The minor groove binder is a dihydropyrroloindole-carboxylate (CDPI3) tripeptide that folds into the minor groove of the target sequence. MGB probes are fitted with an Eclipse Dark Quencher (EDQ) and minor groove binder (MGB) at the 3′ end. If another concentration is desired, please see our Oligonucleotide Resuspension Calculator for resuspension assistance. TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8) is recommended but other molecular biology-grade, nuclease-free diluents may also be used. Re-suspend the dried probes to make stock solutions. For example, 10 nmol of MGB probe is sufficient for 2,500 x 20 uL reactions containing 200 nM probe. Our Reaction Estimator is a useful tool to calculate the number of reactions that can be performed, depending on desired reaction volume and oligonucleotide concentration. MGB probes are available in 10, 20, and 60 nmols delivered. Reaction volumes and number of reactions per vial Multiple freeze-thaw cycles (>10 cycles) should be avoided. Once rehydrated, the oligonucleotides should be aliquoted and stored at -30 ☌ to -15 ☌. MGB probes are shipped at ambient temperature. The Cq values for the Biosearch Technologies and Competitor X manufactured assays were compared for each dilution point and shown to have equivalent performance, with PCR efficiency and R2 values passing PCR criteria (efficiency 90-110% and R2 > 0.98) for all dyes. Assays were run on a BIORAD CFX96 (FAM, TET and CIV/VIC) or an ABI QuantStudio5 (HEX/NED). coli genomic DNA in TaqMan™ Fast Advanced Master Mix, following manufacturers recommendations for assay concentrations and cycling conditions. The performance of each assay was monitored by running a dilution series (1 x 102 to 1 x 108 copies per reaction) of E. All non-labelled oligonucleotides were synthesised by Biosearch Technologies. The probes for each assay were synthesised 5’ FAM, TET, CIV, VIC, HEX or NED and 3’ MGB – Eclipse Dark Quencher (EDQ), by both Biosearch Technologies (blue) and Competitor X (purple). MGB probes manufactured at Biosearch Technologies demonstrate equivalent performance to industry-standard MGB probes.įigure. We meet your regulatory needs from research and development to scale up and commercialisation with ISO 9001 (RUO grade) and ISO 13485/cGMP (diagnostic grade) certified manufacturing options, as applicable.Įquivalent performance to industry standard Manufacturing options under ISO 9001 and ISO 13485 By manufacturing our own dyes, quenchers, specialty amidites, CPG and specialty synthesis columns, we ensure that we are a reliable partner from project inception through commercial scale up. With our unique vertical integration, our chemical manufacturing labs produce most of the critical raw materials used in our own oligo synthesis methods, ensuring a steady and secure supply chain as well as consistent quality at every stage of development. Our multi-site oligo manufacturing operations provide manufacturing redundancy, risk mitigation and surge capacity. This greatly enhances the ∆T m for a mismatch and improves SNP discrimination. Single base mismatches have a greater destabilising effect on MGB probes than other probe types, particularly when the mismatches are within the minor groove binding region.
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