Single-stranded DNA quantitative detection kit as well as preparation method and application thereof
A quantitative detection and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to complete results within 30 minutes, reduce detection accuracy, inaccurate experimental results, etc. The effect of sample detection, fast detection speed and wide linear range
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Embodiment 1
[0027] Example 1: The linear correlation between the detection value of SUPER Green II and the concentration of ssDNA and the determination of the limit of quantification
[0028] 1. Materials and methods
[0029] 1. Preparation of dilution buffer:
[0030] The dilution buffer was 1×TE (pH 7.5) containing 0.002% Tween-20. The preparation method is as follows in Table 1:
[0031] Table 1
[0032] Reagent Volume (mL) 20×TE (pH 7.5) 100 TWEEN-20 0.04 wxya 2 o
to 2000 total: 2000
[0033] According to the above table, accurately measure the corresponding solution and place it in a 2L centrifuge tube, mix and store at 4°C.
[0034] 2. Fluorescent dye SUPER Green II, 10000× diluted with dilution buffer at 1:10000.
[0035] 3. Use dilution buffer as standard 1 for quantitative detection of ssDNA. The synthetic 18bp single-stranded oligonucleotide was added to the dilution buffer to prepare a 100 μM preservation solution. Then it was...
Embodiment 2
[0051] Example 2: Quantitative detection of SUPER Green II circularized ssDNA
[0052] 1. Materials and methods
[0053] 1. Preparation of circularized ssDNA:
[0054] Fragmented DNA was constructed through the MGIEasy Universal DNA Library Prep Kit (Cat. No.: 1000006985) and operated according to the instructions. After the final enzyme digestion and product purification, the obtained is circularized ssDNA.
[0055] 2. SUPER Green II detects the concentration of circularized ssDNA samples:
[0056] Make standard curve: the making of standard curve is the same as 4 in embodiment 1.
[0057] Sample concentration detection: Take 199 μL of SUPER Green II and 1 μL of the sample to be tested and add it to a 0.5mL centrifuge tube dedicated to the Qubit fluorescence detector (the total detection volume is 200 μL), vortex and mix for 2-3 seconds, centrifuge briefly, and place it at room temperature in the dark for two days. Minutes, read the concentration on a Qubit fluorescence d...
Embodiment 3
[0066] Embodiment 3: The stable time that SUPER Green II detects
[0067] 1. Materials and methods
[0068] Standard product fluorescence value RFU stabilization time
[0069] The making of SUPER Green II standard curve is the same as 4 in embodiment 1, then detect the fluorescence value of SUPER Green II combined with standard substance 2. Take 190 μL of SUPER Green II and 10 μL of Standard 2 and add it to a 0.5mL centrifuge tube dedicated to the Qubit fluorescence detector, vortex and mix for 2-3 seconds, centrifuge briefly, and store in the dark at room temperature. After standing for 0min, 30min, 60min, and 120min, respectively, read the fluorescence value RFU of the detection tube on the Qubit detector. In the same way, use The ssDNAAssay Kit detects the fluorescence value RFU of its standard 2 at different storage times.
[0070] SUPER Green II detects the stability time of circularized ssDNA samples
[0071] Three circularized ssDNA samples were used to detect the...
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