DNA adapter for binding magnetic beads and coupling transposase, novel magnetic beads and DOT-seq method
A dot-seq, transposase technology, applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of restricting the development and application of DNA library construction technology, poor uniformity of DNA library, etc. Low DNA quality and input, short time consumption, good library uniformity
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Embodiment 1
[0054] Example 1: Comparison of streptavidin magnetic bead-coupled transposase and carboxyl magnetic bead-coupled transposase.
specific Embodiment approach
[0055] In this example, the yield of DNA library construction was compared between streptavidin magnetic beads coupled with biotin-labeled adapters and carboxyl magnetic beads coupled with amino-labeled adapters (see figure 1 and figure 2 ). The specific implementation is as follows:
[0056] 1) Coupling of streptavidin magnetic beads and biotin-labeled adapters.
[0057] Table 2
[0058] component Dosage 100 µM Biotin-adaptor A 1 µL 100 µM Reverse primer A 3 µL 10× annealing buffer 1 µL H2O 5 µL total capacity 10 µL
[0059] table 3
[0060] component Dosage 100 µM Biotin-adaptor B 1 µL 100 µM Reverse primer A 3 µL 10× annealing buffer 1 µL H2O 5 µL total capacity 10 µL
[0061] 10× annealing buffer contains 500 mM Tris (pH 8.0), 1 M NaCl and 10 mM EDTA.
[0062] 95°C for 5 min, 95°C-15°C (-0.1°C / min), 15°C for 10 min.
[0063] After the reaction, adapter A and adapter B were m...
Embodiment 2
[0091] Example 2: Comparison of amino-modified linear linkers and U-shaped linkers.
[0092] In Example 1, it was verified that carboxyl magnetic beads coupled with amino-modified adapters have higher yields for library construction. In this example, the effects of linear connectors and U-shaped connectors on library yield and library size distribution were verified (see Figure 4 ). The specific implementation is as follows:
[0093] 1) U-shaped joint annealing:
[0094] Table 9
[0095] component Dosage 100 µM U-Amino-adaptor 1 1 µL 100 µM Reverse primer 1 3 µL 100 µM Reverse primer 2 3 µL 10× annealing buffer 2 µL H2O 11 µL total capacity 20 µL
[0096] 95°C for 5 min, 95°C-15°C (-0.1°C / min), 15°C for 10 min. After the annealing was completed, linker magnetic bead coupling and library construction were carried out according to Example 1. The library concentration was measured by Qubit, and the library size distributi...
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