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Body fluid suspension cell DNA extracting kit and extracting method

A suspension cell and kit technology, applied in the field of biology, can solve the problems that the yield and integrity are difficult to meet at the same time, the purpose of extraction cannot be completed, and the concentration of the deproteinized solution is high. The effect of purity

Inactive Publication Date: 2017-06-20
GWP BIOTECHNOLOGIES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The kit includes erythrocyte lysate, deproteinized solution, protein precipitation solution, adjusting binding solution, eluent and proteinase K solution; wherein, the erythrocyte lysate contains 8.29g / L of NH4Cl, 31g / L of KHCO3 and 0.37 g / L of Na2EDTA, pH7.2-7.4; the protein-removing solution is 10% SDS, the protein-removing precipitation solution is saturated sodium chloride solution, the adjusting binding solution is isopropanol, and the eluent It is 70% ethanol, and the concentration of the proteinase K solution is 20 mg / ml, which is the closest prior art of the present application. This method can realize DNA extraction and PCR amplification in blood, but compared with blood, some The composition of body fluids is complex (such as saliva); the amount of suspended cells in some body fluids is very small (such as amniotic fluid), so it is more difficult to meet the three indicators of purity, yield and integrity during the extraction process, and at the same time, the concentration of the deproteinized liquid in this invention is high. , the use of 10% SDS is easy to produce precipitation at low temperature (10°C), and it needs to be heated to dissolve, which is inconvenient to use and high in economic costs. limit

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  • Body fluid suspension cell DNA extracting kit and extracting method
  • Body fluid suspension cell DNA extracting kit and extracting method
  • Body fluid suspension cell DNA extracting kit and extracting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Extraction object: amniotic fluid

[0060] The resuspension liquid is phosphate buffer saline; the lysate is 2mol / L guanidine thiocyanate, 0.1% SDS solution, 0.1% Tween 20 and 5mmol / L flufenzine, and the rest is deionized water ; Proteinase K solution concentration is 10mg / mL; RNase A solution concentration is 10mg / mL; precipitant is 5mol / L potassium chloride solution, washing solution A is absolute ethanol, washing solution B is 71% ethanol, DNA dissolving solution For deionized water, the operation steps are:

[0061] S1: Take 3 mL of amniotic fluid and centrifuge at 2000 rpm for 10 minutes;

[0062] S2: Discard the supernatant, add 2 mL of resuspension solution to resuspend, and centrifuge at 2000 rpm for 10 minutes;

[0063] S3: Discard the supernatant, add 400 μL of lysis solution, 10 μL of proteinase K solution, and 10 μL of RNase A solution, lyse at room temperature for 15 minutes, and then stand at -20°C for 2 minutes;

[0064] S4: Add 100 μL of precipitant, ...

Embodiment 2

[0075] Extraction object: blood

[0076] The resuspension liquid is a mixed solution of 1mmol / L EDTA and 10mmol / L Tris-HCl; the lysate is 8mol / L guanidine hydrochloride and 8v / v% Triton-100, and the rest is deionized water; the concentration of proteinase K solution is 19mg / L mL; the concentration of RNase A solution is 10mg / mL; the precipitant is a mixed solution of 3mol / L potassium chloride and 1mol / L ammonium sulfate, the washing solution A is absolute ethanol, and the washing solution B is 80% ethanol, and the DNA is dissolved Liquid is the mixed solution of the EDTA of 1mmol / L and the Tris-HCl of 100mmol / L;

[0077] The operation steps are:

[0078] S1: Take 100 μL of fresh blood and centrifuge at 13,000 rpm for 1 minute;

[0079] S2: Discard the supernatant, add 1 mL of resuspension solution to resuspend, and centrifuge at 13000 rpm for 1 minute;

[0080] S3: Discard the supernatant, add 200 μL lysate solution, 20 μL proteinase K solution, 20 μL RNase A solution, lyse...

Embodiment 3

[0088] Extraction object: blood

[0089] Wherein the resuspension liquid is normal saline; the lysate is guanidine hydrochloride of 2mol / L, guanidine thiocyanate of 2mol / L, 5% Triton-100, 0.1% Tween 80 and 1mmol / L fluphenidine solution , the rest is deionized water; the concentration of proteinase K solution is 10mg / mL; the concentration of RNase A solution is 20mg / mL; % ethanol, DNA solution is the Tris-HCl solution of 10mmol / L, and operation method comprises the following steps:

[0090] S1: Take 50 μL of fresh blood and centrifuge at 13,000 rpm for 1 minute;

[0091] S2: Discard the supernatant, add 1 mL of resuspension solution to resuspend, and centrifuge at 13000 rpm for 1 minute;

[0092] S3: Discard the supernatant, add 250 μL of lysis solution, 10 μL of proteinase K solution, 10 μL of RNase A solution, lyse at room temperature for 30 minutes, and then stand at -20°C for 2 minutes;

[0093] S4: Add 100 μL of precipitant, centrifuge at 13000 rpm for 5 minutes;

[00...

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Abstract

The invention belongs to the field of biology, and discloses a body fluid suspension cell DNA extracting kit. The extracting kit consists of a resuspension solution, a protease K solution, an RNA enzyme A solution, lysate, a precipitant, a washing solution A, a washing solution B and a DNA dissolved solution. A method for extracting body fluid suspension cell DNA by virtue of the kit comprises the following steps: conducting centrifuging, precipitating and re-suspending, adding the lysate, promoting lysis of the protease K solution, adding the precipitant and conducting centrifuging, adding the washing solution A, conducting centrifuging and washing with the washing solution B, and conducting drying at room temperature and adding the DNA dissolved solution. Different from an existing DNA extracting method or DNA extracting kit which is expensive, simple in object of using and relatively poor in integrity of extracted DNA and which are not applicable to extraction of the body fluid suspension cell DNA, the method and the kit provided by the invention are cheap, relatively high in concentration of the extracted DNA and good in integrity of the extracted DNA and are applicable to extraction of various body fluid suspension cell DNA.

Description

technical field [0001] The invention belongs to the field of biology and relates to a suspension cell DNA extraction kit and an extraction method for extracting body fluids (such as amniotic fluid, blood, saliva, and urine). Background technique [0002] The water contained in the body and various substances dispersed in the water are collectively called body fluids, which can be divided into two parts: intracellular fluid and extracellular fluid. The body fluid in the present invention refers to extracellular fluid; it can be divided into blood, interstitial fluid, lymph fluid, cerebrospinal fluid, urine, saliva, semen, etc., and some special body fluids such as amniotic fluid. [0003] Body fluid suspension cells refer to cells that are free in body fluids, such as free placental cells in amniotic fluid, free epithelial cells in urine, free white blood cells in blood, etc.; these cells reflect the health of the body, so they can be tested Suspended cells in body fluid can...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 赵国栋关淼高珅马勇郑岷雪
Owner GWP BIOTECHNOLOGIES INC
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