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Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit

A kit and magnetic bead method, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of long sample processing time, sample cross-contamination, error-prone extraction steps, etc., to reduce physical injury, save time, and reduce nucleic acid. the effect of the loss

Inactive Publication Date: 2016-03-02
NANJING INST OF ADVANCED LASER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are a variety of whole blood DNA extraction kits that can be used in various fields, but the traditional extraction technology requires precipitation, centrifugation and other operations, and requires a large number of biological samples, resulting in the existence of current whole blood DNA extraction kits for extraction. Many disadvantages, such as complex extraction process, long sample processing time, loss of a large amount of DNA during sample processing, error-prone extraction steps, easy to cause cross-contamination of samples, and the need for multiple instruments, etc.

Method used

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  • Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit
  • Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit
  • Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the preparation of reagent in whole blood genomic DNA extraction kit

[0043] (1) Preparation of cell lysate: first add a small amount of deionized water in the volumetric flask, add guanidine hydrochloride with a concentration of 6mol / L, ethylenediaminetetraacetic acid with a concentration of 10mmol / L, and guanidine hydrochloride with a concentration of 50mmol / L. Trishydroxymethylaminomethane, adding sodium chloride at a concentration of 200mmol / L, adding Triton X-100 at a concentration of 4% (v / v), adding deionized water to the required volume, using sodium hydroxide and hydrochloric acid to adjust the pH value, so that the pH value of the cell lysate is 8.0, and autoclaved for 10 minutes;

[0044] (2) Preparation of the binding solution: first add a small amount of deionized water in the volumetric flask, add polyethylene glycol-8000 with a concentration of 50mmol / L, and sodium chloride with a concentration of 5mol / L, and add deionized water to the requ...

Embodiment 2

[0049] Example 2: Using the kit in Example 1 to extract human whole blood DNA using method one

[0050] Take 8 human whole blood samples, and the specific operation is as follows:

[0051] (1) Take 8 2mLEP tubes, add 100 μL of anticoagulated whole blood, 200 μL of cell lysate, 10 μL of 10 mg / mL proteinase K, and incubate in a 56°C water bath for 30 minutes;

[0052] (2) Then add 20 μL of magnetic beads, 100 μL of binding solution and 300 μL of absolute ethanol, and the binding time is 10 minutes;

[0053] (3) Adsorb the magnetic beads to the tube wall with a magnet, and discard all the liquid in the tube;

[0054] (4) Add 600 μL of Washing Solution I, mix well, use a magnet to absorb the magnetic beads to the tube wall, and discard all the liquid in the tube;

[0055] (5) Add 600 μL of washing solution II, mix well, use a magnet to absorb the magnetic beads to the tube wall, discard all the liquid in the tube, and repeat this step once;

[0056] (6) Add 100 μL of nucleic ac...

Embodiment 3

[0062] Example 3: Using the kit in Example 1 to extract DNA from human whole blood using Method 2

[0063] Take 8 human whole blood samples, and the specific operation is as follows:

[0064] (1) Add 200 μL of cell lysate, 10 μL of 10 mg / mL proteinase K, 200 μL of binding solution and 300 μL of absolute ethanol to row 1 and row 7 of a 96-well reaction plate;

[0065] (2) Add 20 μL of magnetic beads and 600 μL of deionized water to row 2 and row 8 of the 96-well reaction plate;

[0066] (3) Add 600 μL of washing solution I to row 3 and row 9 of the 96-well reaction plate;

[0067] (4) Add 600 μL of washing solution II to the 4-5 row and 10-11 row of the 96-well reaction plate;

[0068] (5) Add 50 μL of nucleic acid eluent to row 6 and row 12 of the 96-well reaction plate;

[0069] (6) Add 100 μL of whole blood to row 1 and row 7 of the 96-well reaction plate, run the nucleic acid extractor, and transfer the nucleic acid eluate in row 6 and row 12 of the 96-well reaction plat...

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Abstract

The invention discloses a whole blood DNA (deoxyribonucleic acid) extraction kit based on a paramagnetic particle method and an application of the extraction kit. The kit comprises a cell lysis solution, a binding solution, a washing solution I, a washing solution II and a nucleic acid eluent. Compared with kits in the prior art, the kit has the advantages of simple and quick operation, use safety, high extraction efficiency, low manufacturing cost and the like; no toxic reagents or organic solvents are used when the kit is used, a blood sample is not required to be pre-treated, the extraction process can be finished manually and can also be automatically finished in a full-automatic nucleic acid extraction instrument, 1-16 animal tissue samples can be treated each time with a 96-hole reaction plate during automatic extraction, other operation is not required in the extraction process, time is saved for whole blood DNA extraction and detection, and the cost is reduced; DNA in the blood can be quickly extracted with the adoption of the kit, the concentration and the purity of extracted nucleic acid are high, the repeatability is good, and the kit can be used for scientific research and clinical detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a whole blood DNA extraction kit based on a magnetic bead method, which can be applied to the whole blood DNA extraction of multiple species. The invention also relates to a method for extracting DNA from whole blood samples using the kit. Background technique [0002] Genomic DNA contains all the genetic information in cells. Extracting DNA from whole blood is a relatively simple method to obtain biological genomic DNA, and it is also an important means and technology for gene correlation research. The quality and yield of DNA extraction directly affect the accuracy of subsequent experiments. [0003] At present, there are a variety of whole blood DNA extraction kits that can be used in various fields, but the traditional extraction technology requires precipitation, centrifugation and other operations, and requires a large number of biological samples, resulting in the existence of ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 孟庆海黄远福王炜
Owner NANJING INST OF ADVANCED LASER TECH
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