Detection method for kanamycin A enzyme-linked aptamer and application of kanamycin A enzyme-linked aptamer
An enzyme-linked aptamer and kanamycin technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the detection method and application of kanamycin A enzyme-linked aptamer Convenience and lack of sensitivity of the method, etc., to achieve good accuracy, easy operation, and strong specificity
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Embodiment 1
[0033] S1. Enzyme-linked aptamer coating.
[0034] Dilute streptavidin with carbonate buffer (pH 9.6, 0.01 mol / L) to obtain 8 μg / mL coating buffer, add the coating buffer to the microplate, 100 μL / well, coat overnight , shake off the liquid in the well, pat dry, add 0.01 mol / L phosphate buffer saline (PBST) containing 0.5% Tween-20 to wash twice, and pat dry.
[0035] The formula of the phosphate buffered saline (PBST) is: Tween-20 500 μL, NaCl 8.5 g, NaCl 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, KH 2 PO 4 0.2g, dilute to 1L with deionized water;
[0036] Add 150 μL / well of blocking buffer (0.1% casein), block in a 37°C water bath for 3 h, shake off the liquid in the wells, pat dry, and dry in a 37°C drying oven for later use.
[0037] S2. The 5' end of the ssDNA aptamer of kanamycin A was modified with biotin (synthesized by Shanghai Sangon Biotechnology Co., Ltd.). The sequence of the ssDNA aptamer of kanamycin A is 5'-TGGGGGTTGAGGCTAAGCCGA-3' (as shown in SEQ ID NO: 1). ...
Embodiment 2
[0045] S1. Enzyme-linked aptamer coating.
[0046] Dilute streptavidin with carbonate buffer (pH 9.6, 0.01 mol / L) to obtain 1 μg / mL coating buffer, add the coating buffer to the microplate, 200 μL / well, and coat overnight , shake off the liquid in the well, pat dry, add 0.01 mol / L phosphate buffer saline (PBST) containing 0.5% Tween-20 to wash twice, and pat dry.
[0047] The formula of the phosphate buffered saline (PBST): same as Example 1.
[0048] Add 150 μL / well of blocking buffer (0.1% casein), block in a 37°C water bath for 3 h, shake off the liquid in the wells, pat dry, and dry in a 37°C drying oven for later use.
[0049] S2. The modified aptamer (the aptamer is the same as in Example 1) was used to contain 5 mM MgCl 2 0.01 mol / L pH 7.4 PBS buffer solution to obtain the aptamer with a concentration of 0.5 nmol / L, add 100 μL to each well, incubate in a water bath at 37 °C for 1 h, wash the plate 5 times, and pat dry to make the aptamer and The streptavidin coat...
Embodiment 3
[0055] S1. Enzyme-linked aptamer coating.
[0056] Dilute streptavidin with carbonate buffer (pH 9.6, 0.01 mol / L) to obtain 32 μg / mL coating buffer, add the coating buffer to the microplate, 100 μL / well, and coat overnight , shake off the liquid in the well, pat dry, add 0.01 mol / L phosphate buffer saline (PBST) containing 0.5% Tween-20 to wash twice, and pat dry.
[0057] The formula of the phosphate buffered saline (PBST): same as Example 1.
[0058] Add 150 μL / well of blocking buffer (0.1% casein), block in a 37°C water bath for 3 h, shake off the liquid in the wells, pat dry, and dry in a 37°C drying oven for later use.
[0059] S2. The aptamer modified with biotin at the 5' end (the aptamer is the same as in Example 1) was treated with 5 mM MgCl 2 0.01 mol / L pH 7.4 PBS buffer to obtain an aptamer solution with a concentration of 4 nmol / L, add 100 μL to each well, incubate in a water bath at 37 °C for 1 h, wash the plate 5 times, and pat dry to make the aptamer Con...
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