Thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof
A thermostable and affinity technology, applied in biochemical equipment and methods, transferases, viruses/phages, etc., can solve the problems of noise, imperfect fidelity, hybrid bands, etc., to avoid primer-dimers, hybrids, etc. Low-band, low-noise effect
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Embodiment 1
[0046] Example 1: Preparation of thermostable Taq enzyme temperature-controlled affinity ligand
[0047] A method for preparing a thermostable Taq enzyme affinity ligand described in this embodiment comprises the following steps:
[0048] 1. Coating and sealing of ELISA plate: Dilute the concentration of Taq enzyme protein to 100 μg / mL with coating buffer, and use it as the coating solution for the first round of panning, and for the second to fourth rounds of panning The concentrations of the coating solution diluted according to the above method are: 50 μg / mL, 25 μg / mL and 10 μg / mL. After the ELISA plate was irradiated by 15W UV lamps at a distance of 10cm for 15mim, the coating solution prepared above was divided into four wells at 100μL / well, and two pretreatment wells were coated with pretreatment well buffer, and placed in a self-sealing Incubate in the bag overnight at 4-8°C, discard the coating solution, wash the plate 4 times with TBST, each time for 3 minutes, pat d...
Embodiment 2
[0058] Example 2: Determination of the Amino Acid Sequence of Taq Enzyme Temperature Control Affinity Ligand
[0059] The phages panned in Example 1 were cloned and amplified, the single-stranded DNA of the phages was dissolved and extracted with sodium iodide, identified by agarose gel electrophoresis and PCR, and then sequenced. According to the reading frame of the phage pIII gene in the coding chain, the amino acid sequence of the foreign polypeptide fused with the pIII protein is deduced to be H-Thr-Trp-Leu-His-Phe-Pro-His-OH, which is the amino acid sequence of the ligand As shown in SEQ ID NO:1.
Embodiment 3
[0060] Embodiment 3: the preparation of whole process hot start Taq enzyme
[0061] Mix the 1000-fold diluted filtrate in Step 10 of Example 1 with 10 μg / μL Taq enzyme in equal volumes to form a full-range hot-start Taq enzyme.
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