Magnetic bead process nucleic acid extraction and transformation kit, and using method thereof
A kit and magnetic bead method technology, which is applied in the field of magnetic bead method nucleic acid extraction and conversion kits, can solve the problems of decreased recovery rate and technical sensitivity, and achieve the effects of short conversion time, saving conversion process time, and improving extraction purity
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Embodiment 1
[0037] Embodiment 1, magnetic bead method nucleic acid extraction conversion kit and its use method
[0038] The magnetic bead method nucleic acid extraction conversion kit includes: lysate, proteinase K, magnetic bead solution, conversion reagent, nucleic acid protection agent, binding solution, washing solution I, washing solution II and eluent. The lysate includes Tris-HCl, guanidine isothiocyanate, sodium hydroxide, Nonidet P 40, sodium azide, isopropanol, Triton X-100, catechin and Carrier RNA. The magnetic bead solution includes Tris-HCl, EDTA-2Na and magnetic beads. The content of the magnetic beads in the magnetic bead liquid is 50mg / ml, and the magnetic beads are nano-magnetic particles made of supercis ferric oxide or supercis iron sesquioxide, and the surface is modified with hydroxyl or carboxyl Silica coated. Described transformation reagent comprises bisulfite and / or bisulfite, and urea and hydroquinone; Described bisulfite comprises sodium bisulfite, and descr...
Embodiment 2
[0048] Embodiment 2, conversion step optimization
[0049] Take 6 identical samples and divide them into two groups of A and B respectively, the samples of group A are transformed by the kit described in Example 1, the samples of group B are subjected to nucleic acid extraction according to the steps of the kit of the present invention, and conventional transformation is added in the transformation step The desulfo treatment step conversion in the flow process is to add a strong base and incubate at room temperature for 15 minutes after the primary washing to perform the desulfo treatment, and the other steps are the same as the conversion steps in the kit of the present invention. The two sets of nucleic acid samples after transformation are detected according to the following steps:
[0050] a) Mix the following reagents in a PCR tube:
[0051] 2×PCR Mix
20μl
taq enzyme
0.5μl
transforming DNA solution
10μl
NF water
9.5μl
...
Embodiment 3
[0055] Example 3, Effects of Different Transformation Temperatures and Transformation Times on the Transformation Effect of Nucleic Acid Samples
[0056] By setting the conversion temperature and conversion time of different groups (see Table 1 below for details), the rest of the steps were performed using the method in Example 1 to perform nucleic acid extraction and conversion on the same batch of samples. Twelve groups obtained DNA (referred to as A1-A4, B1-B4, and C1-C4 in sequence) through different transformation temperatures and transformation time methods for methylation detection by real-time fluorescent PCR. The detection results are shown in Table 2.
[0057] Table 1 Transformation condition settings of different experimental groups
[0058]
[0059] Table 2 Methylated DNA detection results after treatment with different experimental groups transformation conditions
[0060]
[0061] It can be seen that in different transformation temperature methods, using r...
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