CN117165575A - Kit for automatically extracting dry blood spot DNA based on nano magnetic beads - Google Patents
Kit for automatically extracting dry blood spot DNA based on nano magnetic beads Download PDFInfo
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- CN117165575A CN117165575A CN202311093777.2A CN202311093777A CN117165575A CN 117165575 A CN117165575 A CN 117165575A CN 202311093777 A CN202311093777 A CN 202311093777A CN 117165575 A CN117165575 A CN 117165575A
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- concentration
- kit
- magnetic beads
- aminomethane
- automatically extracting
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- 239000011324 bead Substances 0.000 title claims abstract description 38
- 210000004369 blood Anatomy 0.000 title claims abstract description 33
- 239000008280 blood Substances 0.000 title claims abstract description 33
- 238000005406 washing Methods 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000009089 cytolysis Effects 0.000 claims abstract description 8
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 19
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000001509 sodium citrate Substances 0.000 claims description 12
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 10
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 9
- 239000006166 lysate Substances 0.000 claims description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 239000013504 Triton X-100 Substances 0.000 claims description 6
- 229920004890 Triton X-100 Polymers 0.000 claims description 6
- 238000000197 pyrolysis Methods 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 abstract description 15
- 238000000034 method Methods 0.000 abstract description 14
- 102000039446 nucleic acids Human genes 0.000 abstract description 14
- 108020004707 nucleic acids Proteins 0.000 abstract description 14
- 150000007523 nucleic acids Chemical class 0.000 abstract description 14
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 6
- 238000001556 precipitation Methods 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 238000007885 magnetic separation Methods 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- 102000053602 DNA Human genes 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 12
- 229960001484 edetic acid Drugs 0.000 description 12
- 238000005336 cracking Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000002205 phenol-chloroform extraction Methods 0.000 description 2
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical class ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000409 membrane extraction Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for automatically extracting DNA of a dried blood spot based on nano magnetic beads, belonging to the technical field of molecular biology. Comprises a lysis solution AL, proteinase K, a lysis binding solution BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluting solution DE. The invention provides a kit for automatically extracting DNA of dried blood spots based on nano magnetic beads, which is based on a nano magnetic bead adsorption nucleic acid technology, purifies high-quality nucleic acid from a dried blood spot sample, does not contain toxic components such as phenol and chloroform, does not need time-consuming alcohol precipitation, can perform high-flux extraction experiments by integrating a magnetic rod method or a pipetting automatic nucleic acid extractor, can protect the safety of experimental staff while improving the extraction efficiency, and can be manually operated by matching a magnetic separation frame, and is compatible with the automatic method and the manual method for extraction.
Description
Technical Field
The invention relates to a kit for automatically extracting DNA of a dried blood spot based on nano magnetic beads, belonging to the technical field of molecular biology.
Background
The extraction of genomic nucleic acid from dried blood spots is a very common requirement, and the extraction of the genome of lymphocytes in dried blood spots can be applied to clinical medical disease diagnosis and adjuvant therapy through the analysis of the genome. At present, the dry blood spot genome DNA extraction mainly comprises a column membrane method and a magnetic bead method, and compared with a saturated phenol-chloroform method, a salting-out precipitation method and the like, the magnetic bead method does not need toxic phenol-chloroform extraction or time-consuming alcohol precipitation in the extraction process, so that the safety of operators is ensured; furthermore, the column membrane extraction cannot be used in machines nor in high throughput operation by automated methods, and the extraction efficiency is general.
Disclosure of Invention
In view of the above, the invention provides a kit for automatically extracting dry blood spot DNA based on nano magnetic beads, which is based on a nano magnetic bead adsorption nucleic acid technology, purifies high-quality nucleic acid from a dry blood spot sample, does not contain toxic components such as phenol and chloroform, does not need time-consuming alcohol precipitation, can perform high-flux extraction experiments by integrating a magnetic rod method or a pipetting method automatic nucleic acid extractor, can protect the safety of experimental staff while improving the extraction efficiency, and can be manually operated by matching a magnetic separation frame, and is compatible with the automatic method and the manual method.
The invention provides a kit for automatically extracting dry blood spot DNA (deoxyribonucleic acid) based on nano magnetic beads, which comprises a lysate AL, proteinase K, a lysate BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluent DE;
the lysate AL comprises tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate;
the lysis binding solution BLN comprises tris (hydroxymethyl) aminomethane, guanidine thiocyanate, sodium citrate and triton X-100;
the magnetic beads MBVB are polystyrene coated silicon hydroxyl magnetic beads;
the washing liquid DW1 comprises guanidine thiocyanate, ethylenediamine tetraacetic acid, sodium citrate, sodium chloride and absolute ethyl alcohol;
the washing liquid DW2 comprises tris (hydroxymethyl) aminomethane, sodium chloride and absolute ethyl alcohol;
the eluent DE comprises tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the pyrolysis liquid AL is 20-80mmol/L, the concentration of the ethylenediamine tetraacetic acid is 5-30mmol/L, the concentration of the sodium chloride is 50-300mmol/L, and the concentration of the sodium dodecyl sulfate is 0.1% -2%.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the lysis binding solution BLN is 20-80mmol/L, the concentration of guanidine thiocyanate is 2.5-5mol/L, the concentration of sodium citrate is 0.1-2%, the concentration of triton X-100 is 5-15%, and HCL and NaOH are used for adjusting the PH to be 5-10.
Preferably, the concentration of ethylenediamine tetraacetic acid in the washing solution DW1 is 1-10mmol/L, the concentration of sodium citrate is 0.1-2%, the concentration of sodium chloride is 10-200mmol/L, and the concentration of absolute ethyl alcohol is 30-80%.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the washing solution DW2 is 20-80mmol/L, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30% -80%.
Preferably, the concentration of the tris (hydroxymethyl) aminomethane in the eluent DE is 10-50mmol/L, the concentration of the ethylenediamine tetraacetic acid is 1-10mmol/L, and HCL and NaOH are used for adjusting the PH to be 5-10.
Preferably, the kit also comprises blood sheets and centrifuge tubes.
The invention has the beneficial effects that:
the invention provides a kit for automatically extracting dry blood spot DNA (deoxyribonucleic acid) based on a nano magnetic bead adsorption nucleic acid technology, which is used for purifying high-quality nucleic acid from a dry blood spot sample, does not contain toxic components such as phenol and chloroform, does not need time-consuming alcohol precipitation, can perform high-flux extraction experiments by integrating a magnetic rod method or a pipetting automatic nucleic acid extractor, can realize automatic and high-flux operation, can simultaneously realize treatment of a plurality of samples in the previous extraction time of one sample by matching with the 96/192/384-hole automatic nucleic acid extractor, meets the requirement of the current biological high-flux operation, can protect the safety of experimental staff while improving the extraction efficiency, and can perform manual operation by matching with a magnetic separation frame, and is compatible with the automatic and manual extraction.
Drawings
FIG. 1 is an agarose gel diagram of a kit for automatically extracting DNA of dried blood spots based on nano magnetic beads.
Detailed Description
Preferred embodiments of the present invention will be described in detail below.
The invention provides a kit for automatically extracting dry blood spot DNA (deoxyribonucleic acid) based on nano magnetic beads, which comprises a lysate AL, proteinase K, a lysate BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluent DE;
the pyrolysis liquid AL comprises tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate, wherein the concentration of the tris (hydroxymethyl) aminomethane in the pyrolysis liquid AL is 20-80mmol/L, the concentration of the ethylenediamine tetraacetic acid is 5-30mmol/L, the concentration of the sodium chloride is 50-300mmol/L, and the concentration of the sodium dodecyl sulfate is 0.1% -2%;
the cracking combination solution BLN comprises tris (hydroxymethyl) aminomethane, guanidine thiocyanate, sodium citrate and triton X-100, wherein the concentration of the tris (hydroxymethyl) aminomethane in the cracking combination solution BLN is 20-80mmol/L, the concentration of the guanidine thiocyanate is 2.5-5mol/L, the concentration of the sodium citrate is 0.1-2%, the concentration of the triton X-100 is 5-15%, and HCL and NaOH are used for adjusting the PH to be 5-10;
the magnetic beads MBVB are polystyrene coated silicon hydroxyl magnetic beads;
the washing liquid DW1 comprises guanidine thiocyanate, ethylene diamine tetraacetic acid, sodium citrate, sodium chloride and absolute ethyl alcohol, wherein the concentration of the ethylene diamine tetraacetic acid in the washing liquid DW1 is 1-10mmol/L, the concentration of the sodium citrate is 0.1-2%, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30-80%;
the washing solution DW2 comprises tris (hydroxymethyl) aminomethane, sodium chloride and absolute ethyl alcohol, wherein the concentration of the tris (hydroxymethyl) aminomethane in the washing solution DW2 is 20-80mmol/L, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30-80%;
the eluent DE comprises tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid, wherein the concentration of the tris (hydroxymethyl) aminomethane in the eluent DE is 10-50mmol/L, the concentration of the ethylenediamine tetraacetic acid is 1-10mmol/L, and HCL and NaOH are used for adjusting the PH to 5-10.
The invention also comprises a blood tablet and a centrifuge tube.
The application method of the invention comprises the following steps:
1) Punching 3-5 blood slices with the diameter of 3mm on the FTA Card or the blood slices by using a puncher or cutting the blood slices, and transferring the blood slices into a 1.5mL or 2.0mL centrifuge tube;
2) Adding 400 mu L of lysate AL and 20 mu L of proteinase K into the centrifuge tube, mixing for 30 seconds by high-speed vortex on a vortex instrument, and checking to ensure that blood chips are immersed in the solution;
3) Oscillating the temperature bath at 60 ℃ for 45 minutes, taking out vortex 1 time every 15 minutes and 30 seconds each time during the temperature bath if the thermostat has no oscillation function;
4) After returning to room temperature, centrifuging to collect the liquid drops of the pipe cover into the pipe;
5) Transferring all the warm bath liquid in the step 4) into a pre-assembled plate cracking binding liquid BLN hole, completing extraction by a nucleic acid extractor operation program, and respectively taking 2ul of the extracted liquid, and measuring the concentration and 260/280,260/230 ratio by a nanodrop spectrophotometer; 2ul of each is taken and subjected to agarose gel electrophoresis to verify DNA extraction;
6) Nucleic acid extractor program settings
| Step one | The lysate BLN was mixed with the sample for 10 minutes |
| Step two | Magnetic bead |
| Step three | Mixing nucleic acid at room temperature for 5 min, and adsorbing magnetic beads |
| Step four | Washing liquid DW1 is washed for 1 time, and magnetic beads are adsorbed |
| Step five | Washing liquid DW2 washes 1 time, and adsorbs magnetic beads |
| Step six | Washing liquid DW2 washes 1 time, and adsorbs magnetic beads |
| Step seven | Airing for one minute |
| Step eight | Eluting with 70 ℃ eluent DE for 5 minutes to adsorb magnetic beads |
| For a long time | Magnetic bead |
7) Results display
Nanodrop spectrophotometer for measuring concentration and 260/280,260/230 ratio
The numbers 1-5 are the genomic DNA of the dried blood spots extracted by the commercial A brand column method.
The number 6-10 is the extraction of the genomic DNA of the dried blood spots by the kit of the invention.
The invention and its embodiments have been described above without limitation, and the actual construction is not limited thereto. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (7)
1. A kit for automatically extracting dry blood spot DNA based on nano magnetic beads is characterized in that: comprises a lysis solution AL, proteinase K, a lysis binding solution BLN, magnetic beads MBVB, a washing solution DW1, a washing solution DW2 and an eluent DE;
the lysate AL comprises tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate;
the lysis binding solution BLN comprises tris (hydroxymethyl) aminomethane, guanidine thiocyanate, sodium citrate and triton X-100; the magnetic beads MBVB are polystyrene coated silicon hydroxyl magnetic beads;
the washing liquid DW1 comprises guanidine thiocyanate, ethylenediamine tetraacetic acid, sodium citrate, sodium chloride and absolute ethyl alcohol; the washing liquid DW2 comprises tris (hydroxymethyl) aminomethane, sodium chloride and absolute ethyl alcohol;
the eluent DE comprises tris (hydroxymethyl) aminomethane and ethylenediamine tetraacetic acid.
2. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the tris (hydroxymethyl) aminomethane in the pyrolysis liquid AL is 20-80mmol/L, the concentration of the ethylenediamine tetraacetic acid is 5-30mmol/L, the concentration of the sodium chloride is 50-300mmol/L, and the concentration of the sodium dodecyl sulfate is 0.1% -2%.
3. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the tris (hydroxymethyl) aminomethane in the lysis binding solution BLN is 20-80mmol/L, the concentration of guanidine thiocyanate is 2.5-5mol/L, the concentration of sodium citrate is 0.1-2%, the concentration of triton X-100 is 5-15%, and HCL and NaOH are used for adjusting the PH to be 5-10.
4. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of ethylenediamine tetraacetic acid in the washing liquid DW1 is 1-10mmol/L, the concentration of sodium citrate is 0.1% -2%, the concentration of sodium chloride is 10-200mmol/L, and the concentration of absolute ethyl alcohol is 30% -80%.
5. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the tris (hydroxymethyl) aminomethane in the washing liquid DW2 is 20-80mmol/L, the concentration of the sodium chloride is 10-200mmol/L, and the concentration of the absolute ethyl alcohol is 30% -80%.
6. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the concentration of the trimethylol aminomethane in the eluent DE is 10-50mmol/L, the concentration of the ethylenediamine tetraacetic acid is 1-10mmol/L, and the PH is adjusted to 5-10 by using HCL and NaOH.
7. The kit for automatically extracting dry blood spot DNA based on the nano magnetic beads according to claim 1, wherein the kit comprises the following components: the utility model also comprises a blood tablet and a centrifuge tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311093777.2A CN117165575A (en) | 2023-08-29 | 2023-08-29 | Kit for automatically extracting dry blood spot DNA based on nano magnetic beads |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311093777.2A CN117165575A (en) | 2023-08-29 | 2023-08-29 | Kit for automatically extracting dry blood spot DNA based on nano magnetic beads |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117165575A true CN117165575A (en) | 2023-12-05 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311093777.2A Pending CN117165575A (en) | 2023-08-29 | 2023-08-29 | Kit for automatically extracting dry blood spot DNA based on nano magnetic beads |
Country Status (1)
| Country | Link |
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| CN (1) | CN117165575A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118389492A (en) * | 2024-07-01 | 2024-07-26 | 广州凯普医药科技有限公司 | Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof |
-
2023
- 2023-08-29 CN CN202311093777.2A patent/CN117165575A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118389492A (en) * | 2024-07-01 | 2024-07-26 | 广州凯普医药科技有限公司 | Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof |
| CN118389492B (en) * | 2024-07-01 | 2024-10-15 | 广州凯普医药科技有限公司 | Kit for extracting genomic DNA of dried blood spots by magnetic bead method and application thereof |
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