CN101619289B - Device for enriching and purifying long-fragment nucleic acid - Google Patents
Device for enriching and purifying long-fragment nucleic acid Download PDFInfo
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- CN101619289B CN101619289B CN 200810029118 CN200810029118A CN101619289B CN 101619289 B CN101619289 B CN 101619289B CN 200810029118 CN200810029118 CN 200810029118 CN 200810029118 A CN200810029118 A CN 200810029118A CN 101619289 B CN101619289 B CN 101619289B
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- nucleic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/06—Tubular
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/12—Purification
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Abstract
The invention discloses a device for enriching and purifying long-fragment nucleic acid, which comprises a cylindrical main body 1, wherein open ports at two ends of the cylindrical main body 1 are coated with semi-transparent films 2, the cylindrical main body is provided with a liquid taking hole 3 and a loading slot 4, an agarose gel is filled in part of cylindrical main body below the loading slot, the part of the cylindrical main body below the liquid taking hole is kept as a cavity and is used for infusing an electrophoretic buffer. The device effectively purifies and enriches the long-segment nucleic acid by using the difference of the mobility of non-amphiprotic impurities under an electric field condition and amphiprotic interferents with different molecular sizes under the electric field condition.
Description
Technical field
The present invention relates to a kind of device for enriching and purifying long-fragment nucleic acid.
Background technology
The foundation of nucleic acid extraction technology is the key that ensures that respective downstream experiment such as PCR is carried out smoothly; Up to the present, disturb impurity less, preserve better, leaching process that nucleic acid extraction technology that the biomaterial of processing or part deep processing basically all can be accomplished to utilize biological technical field to know (common nucleic acid extracting reagent or utilize commercially available special device like purification column method, paramagnetic particle method, ultrafiltration centrifuging etc.) is accomplished the nucleic acid that is suitable for the downstream experiment smoothly.And interference impurity is many or deep processing, preservation are not good at, the time is after the extraction of the biomaterial amplifying nucleic acid that nucleic acid is degraded in a large number, effective nucleic acid content is extremely low such as corruption for a long time even highly; Then be all still unsolved difficult problem of at present domestic and international Molecular Biology Lab, so that many research work are got into a difficult position.Setting up the nucleic acid extraction technology to such biomaterial of practical easy handling, to fill up the blank of molecular biology experiment technology for many years, is the urgent need of this area research work.
Summary of the invention
The objective of the invention is to develop a kind of device for enriching and purifying long-fragment nucleic acid, to solve the problem that prior art exists.
In order to achieve the above object; The device for enriching and purifying long-fragment nucleic acid of this development work invention comprises a cylindrical body 1, coats semi-permeable membranes 2 at the both ends open port; On cylindrical body, be provided with liquid outlet 3 and loading slot 4; Be filled with sepharose in the part cylindrical body below loading slot, and the part cylindrical body of liquid outlet below remains cavity, as injecting electrophoretic buffer.
The concrete working method of said apparatus is following:
(a) electrophoresis chamber adds electrophoretic buffer;
(b) cavity of the part cylindrical body of liquid outlet below is filled it up with electrophoretic buffer, and covers tight liquid outlet plug;
(c) with in the electrophoretic buffer in the nucleic acid enriching purification devices immersion electrophoresis chamber;
(d) behind nucleic acid to be purified and sample-loading buffer and the indicating dye mixing, join loading slot, and energising;
(e) treat that the indicating dye band all gets in the damping fluid of liquid outlet below, discards the damping fluid of liquid outlet below;
(f) refill the cavity of electrophoretic buffer in the liquid outlet below, cover tight liquid outlet plug, be reentered in the electrophoresis chamber, continue the energising electrophoresis, the damping fluid of collecting the liquid outlet lower cavity carries out phenol/chloroform extracting.
This device is utilized under the current field condition, the impurity of non-amphoteric properties and the difference of the mobility of the different amphoteric properties obscurant of molecular size, purifying and enrichment long-fragment nucleic acid effectively under current field condition.
Below will through this particularly embodiment come the present invention is carried out detailed description.
Description of drawings
Fig. 1 is the structural representation of described device for enriching and purifying long-fragment nucleic acid
Embodiment
As shown in Figure 1, be the structural representation of described device for enriching and purifying long-fragment nucleic acid.This device comprises a horizontal transparent cylindrical body 1, coats semi-permeable membranes 2 at the both ends open port through sealer lid 8, and sealer lid 8 can let semi-permeable membranes 2 be fixed on the port of cylindrical body with openable mode.Be provided with liquid outlet 3 and loading slot 4 in the cylindrical body side wall upper part, thief hole 3 has plug 5.Be filled with sepharose 6 in the part cylindrical body below loading slot, and the part cylindrical body of liquid outlet below remains cavity 7, as injecting electrophoretic buffer.
Concrete operation instructions is following:
(a) electrophoresis chamber adds electrophoretic buffer;
(b) open nucleic acid enriching purification devices liquid outlet plug 5, add the electrophoresis damping fluid in liquid outlet,, cover tight liquid outlet plug to filling it up with disposable dropper;
(c) with in the electrophoretic buffer in the nucleic acid enriching purification devices immersion electrophoresis chamber;
(d) behind nucleic acid to be purified and sample-loading buffer (the containing indicating dye) mixing, join loading slot, opening power is pressed 10V/cm adjustment voltage, and energising; The molecular weight of the molecular weight ratio target nucleic acid of indicating dye is slightly little.
(e) treat that the indicating dye band all gets in the damping fluid of liquid outlet below (even continuing electrophoresis 30-60min); Take off device for enriching and purifying from electrophoresis chamber; Open the liquid outlet plug; With whole liquid in another disposable dropper sucking-off liquid outlet, add a small amount of electrophoretic buffer in liquid outlet, careful repeatedly suction flushing back sucking-off; When the indicating dye bar all gets in the damping fluid of liquid outlet below; Show that the little impurity such as nucleic acid of molecular weight of molecular weight ratio dyestuff all enter into the damping fluid of liquid outlet below from gel; And owing to holding back of semi-permeable membranes; These impurity rest in the damping fluid of liquid outlet below, discard this part damping fluid, have then removed the little impurity of molecular weight ratio purpose nucleic acid.
(f) refill electrophoretic buffer in liquid outlet, cover tight liquid outlet plug, be reentered in the electrophoresis chamber, continue energising, after about 5-6 hour (but proper extension time), powered-down;
(g) open the liquid outlet plug; With another clean whole liquid in disposable dropper sucking-off liquid outlet below; In a 1.5mL or 2mL centrifuge tube; Add a small amount of electrophoretic buffer in liquid outlet, careful repeatedly suction flushing back sucking-off also is transferred to this centrifuge tube equally, controls TV in 1.6mL as far as possible;
The liquid of mixing above-mentioned (g) collection step adopts phenol/chloroform method extracting once more.Concrete grammar is: add equal-volume phenol/chloroform/primary isoamyl alcohol, fully shake about 30s, the centrifugal 10min of 7500 * g room temperature; The careful supernatant of drawing adds 3M NaAc to another 1.5mL centrifuge tube by 1/10 volume, and mixing adds LA solution, mixing; Add the equal-volume Virahol, mixing ,-20 ℃ leave standstill about 40min-60min after, 27000-30000 * g; 4 ℃ of centrifugal 10min abandon or adopt supernatant, and deposition adds 500 μ L 70%-80% precooled ethanol, and fully the turned upside down mixing is with washing and precipitating; 27000-30000 * g, 4 ℃ of centrifugal 5min, supernatant discarded, it is dry to put nucleic acid vacuum-drying appearance.
Get and be preheated to about 80-90 ℃ of TE damping fluid 30-50 μ L and fully dissolve dry extract, this is the nucleic acid purification product.
Claims (5)
1. device for enriching and purifying long-fragment nucleic acid; It is characterized in that: comprise a cylindrical body (1); Coat semi-permeable membranes (2) at the both ends open port, on cylindrical body, be provided with liquid outlet (3) and loading slot (4), be filled with sepharose (6) in the part cylindrical body below loading slot; And the part cylindrical body of liquid outlet below remains cavity (7), as injecting electrophoretic buffer.
2. by the described device for enriching and purifying long-fragment nucleic acid of claim 1, it is characterized in that: described liquid outlet (3) has a plug (5).
3. by the described device for enriching and purifying long-fragment nucleic acid of claim 1, it is characterized in that: described semi-permeable membranes (2) is fixed on the port part of cylindrical body through the sealer lid.
4. by the described device for enriching and purifying long-fragment nucleic acid of claim 1, it is characterized in that: can be monomer or series, parallel polymer.
5. by the described device for enriching and purifying long-fragment nucleic acid of claim 1, it is characterized in that: described cylindrical body is processed by transparent material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810029118 CN101619289B (en) | 2008-06-30 | 2008-06-30 | Device for enriching and purifying long-fragment nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810029118 CN101619289B (en) | 2008-06-30 | 2008-06-30 | Device for enriching and purifying long-fragment nucleic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101619289A CN101619289A (en) | 2010-01-06 |
| CN101619289B true CN101619289B (en) | 2012-04-25 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200810029118 Expired - Fee Related CN101619289B (en) | 2008-06-30 | 2008-06-30 | Device for enriching and purifying long-fragment nucleic acid |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002085048A (en) * | 2000-09-11 | 2002-03-26 | Hitachi Ltd | Apparatus for purifying nucleic acid |
| JP2004113043A (en) * | 2002-09-25 | 2004-04-15 | Fuji Photo Film Co Ltd | Nucleic acid-separating and purifying device |
| JP2005110503A (en) * | 2003-10-02 | 2005-04-28 | Arkray Inc | Method for purifying nucleic acid and device therefor |
| CN1990497A (en) * | 2002-11-28 | 2007-07-04 | 爱科来株式会社 | Method and apparatus for concentration and purification of nucleic acid |
-
2008
- 2008-06-30 CN CN 200810029118 patent/CN101619289B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002085048A (en) * | 2000-09-11 | 2002-03-26 | Hitachi Ltd | Apparatus for purifying nucleic acid |
| JP2004113043A (en) * | 2002-09-25 | 2004-04-15 | Fuji Photo Film Co Ltd | Nucleic acid-separating and purifying device |
| CN1990497A (en) * | 2002-11-28 | 2007-07-04 | 爱科来株式会社 | Method and apparatus for concentration and purification of nucleic acid |
| JP2005110503A (en) * | 2003-10-02 | 2005-04-28 | Arkray Inc | Method for purifying nucleic acid and device therefor |
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| CN101619289A (en) | 2010-01-06 |
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