CN101587237B - Method for cleaning and reusing microdissection sample-collecting tubes - Google Patents
Method for cleaning and reusing microdissection sample-collecting tubes Download PDFInfo
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- CN101587237B CN101587237B CN2009100121556A CN200910012155A CN101587237B CN 101587237 B CN101587237 B CN 101587237B CN 2009100121556 A CN2009100121556 A CN 2009100121556A CN 200910012155 A CN200910012155 A CN 200910012155A CN 101587237 B CN101587237 B CN 101587237B
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Abstract
The invention discloses a method for cleaning and reusing microdissection sample-collecting tubes. The method can be implemented according to the following steps in turn: (1) adding xylene to a collecting tube, capping and oscillating the collecting tube and discarding the xylene; (2) adding suspension A to the collecting tube, oscillating and then emptying the collecting tube; (3) adding double-distilled water to the collecting tube while adding silica particles, oscillating and then emptying the collecting tube; (4) repeating the step (2) and the step (3) twice in turn; (5) adopting the double-distilled water to wash the collecting tube; (6) uncapping the collecting tube and placing the collecting tube in a refrigerator till liquid completely evaporates; (7) taking the collecting tube out, smearing polylysine on the surface of a viscous module on the inner surface of a tube cap, putting the collecting tube back into the refrigerator and keeping the collecting tube standing; and (8) repeating the step (7). The method can effectively maintain and repair the adhesion property of the viscous module on the inner surface of the tube cap of the collecting tube, and can effectively remove superfluous substances remaining in the collecting tube so as to repeatedly recycle the collecting tube and save a large amount of experiment funding.
Description
Technical field
The present invention relates to micro-dissections sample consumptive material cleaning technique field, a kind of specifically microdissection sample-collecting tubes cleans the method for utilizing again.
Background technology
The appearance of laser capture micro-dissections (Laser Capture Microdissection) technology and system thereof, make the related scientific research worker can accurately separate purpose tissue specimen to be studied, cell (living cells of in vitro culture and be fixed on histocyte in the section), organelle even chromosomal region band, be used for follow-up study, the accuracy of its control sample is subjected to the favor of whole world researcher, and at present most strong research institutions all have been equipped with this technology and related facility.
When carrying out micro-dissections with histotomy, need use a kind of consumptive material of special use, this consumptive material is a tubular container (hereinafter to be referred as collection tube) with cover, it is characterized by on the inside surface of collection tube lid and carry ganoid special stickiness module, the film that has destination organization that is separated by cut on the film microslide when utilizing the stickiness absorption affinity of module to collect micro-dissections.This collection tube consumptive material price is very expensive; Simultaneously, because the purpose sample that is used to study needs the enough quantity of enrichment, make that this consumptive material usage quantity is bigger in the research process, with regard to single sample, the gene level research that DNA, RNA are relevant generally needs 2-3, protein level in addition need dozens of.At present, domestic still do not have manufacturer can produce this special-purpose consumptive material, reuses this consumptive material good method of can yet be regarded as.Because laser can burn stickiness module surface and cause the burnt trace of appearance in the experimentation, thereby reduces its adhesion; Simultaneously, tissue, nucleic acid, albumen and other small-molecule substances that is bonded on the lid can pollute in follow-up molecular biology experiment.Therefore, how can effectively remove tissue, nucleic acid, albumen and other small-molecule substances that the collection tube pipe covers, do not damage the adhesion that the collection tube pipe covers special stickiness module again, and can repair again stickiness module surface in experimentation by the burnt trace of laser burn, be the recycling this collection tube a difficult problem.Conventional mechanical washing, High Temperature High Pressure washing, strong acid and strong base washing etc. all can damage the adhesion of collection tube, and ultraviolet ray, cobalt 60 irradiation also can only the deactivation microorganisms, can not remove small-molecule substance.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art part and a kind of collection tube pipe interior surface stickiness module adhesion that can effectively keep and repair is provided, and can effectively remove the tissue, nucleic acid, albumen and other small-molecule substances that remain in the collection tube, thereby collection tube is repeatedly recycled, and the microdissection sample-collecting tubes of saving a large amount of experiment fees cleans the method for utilizing again.
For achieving the above object, the present invention is achieved in that
Microdissection sample-collecting tubes cleans and utilizes method again, can implement successively as follows:
(1) in collection tube, adds the dimethylbenzene that accounts for collection tube volume 1/2～2/3, cover collection tube pipe lid, and vibrated 2～5 minutes, open the pipe lid, discard dimethylbenzene;
(2) in the described collection tube of step (1), add the suspension A that accounts for collection tube volume 1/2～2/3, the lid upper tube cap, and vibrated 2～3 minutes, open the pipe lid, the turned letter collection tube;
(3) in the described collection tube of step (2), add the distilled water that accounts for collection tube volume 1/2～2/3, add the silica dioxide granule of 50mg diameter simultaneously less than 0.1mm, the lid upper tube cap, and vibrated 2～3 minutes, open the pipe lid, the turned letter collection tube;
(4) repeating step (2) and step (3) twice successively;
(5) adopt distilled water that step (4) gained collection tube was washed 3～5 minutes;
(6) collection tube is uncapped, insert in 2～8 ℃ of refrigerators, evaporate fully until collection tube lid and liquid in pipe;
(7) take out collection tube, in super-clean bench, smear concentration toward collection tube pipe interior surface stickiness module surface and be 4～8% poly-D-lysine, rapidly collection tube is put back to refrigerator, leave standstill;
(8) repeating step (7) once.
As a kind of preferred version, suspension A of the present invention is mixed by hydrochloric acid, alcohol and EDTA, and adding 50mg diameter is made less than the silica dioxide granule of 0.1mm.
As another kind of preferred version, the concentration of hydrochloric acid of the present invention is 1.5%; The concentration of described alcohol is 30%; The concentration of described EDTA is 0.25mol/L.
Further, the volume ratio of hydrochloric acid of the present invention, alcohol and EDTA is 0.5～1.5: (96.5～99): 0.5～2.
Further, time of repose t 〉=24h in the step of the present invention (7).
The present invention compared with prior art has following characteristics:
1, do not relate to damage violence, High Temperature High Pressure, strong acid-base etc. in the whole operation flow process of the present invention to the prejudicial factor of film;
2, the present invention is fully cleaned the stickiness module, the adhesion of the module that adequately protected simultaneously by using suspension A;
3, the present invention can well repair the burning trace that laser causes on stickiness module surface by smearing poly-D-lysine toward the surface, and recovers to burn the adhesion at trace position to a certain extent;
4, the collection tube after the present invention cleans does not contain testing influential composition;
5, reagent required for the present invention is the conventional reagent of biology laboratory, and cheap, but batch operation;
6, recycling rate of waterused height of the present invention can be saved 10-20 collection tube quantity doubly, saves a large amount of research fundings.
Description of drawings
The invention will be further described below in conjunction with the drawings and specific embodiments.Protection scope of the present invention not only is confined to the statement of following content.
Whether Fig. 1 cleans up 2% the agarose electrophoresis figure that carries out for the checking collection tube.
Embodiment
Microdissection sample-collecting tubes cleans and utilizes method again, can implement successively as follows:
(1) in collection tube, add the dimethylbenzene that accounts for collection tube volume 1/2, the lid upper tube cap, the pipe lid is opened in vortice vibration 3 minutes, discards dimethylbenzene;
(2) in collection tube, add the suspension A (mixed in 1: 97: 1 by volume by 1.5% hydrochloric acid, 30% alcohol, 0.25mol/L EDTA, and adding 50mg diameter being formulated less than the silica dioxide granule of 0.1mm) that accounts for collection tube volume 2/3.The lid upper tube cap, the pipe lid is opened in vortice vibration 2.5 minutes, the turned letter collection tube;
(3) in collection tube, add the distilled water that accounts for collection tube volume 1/2, add the silica dioxide granule of 50mg diameter simultaneously less than 0.1mm, the lid upper tube cap, the pipe lid is opened in vortice vibration 2.5 minutes, the turned letter collection tube;
(4) repeating step (2) and step are (3) twice;
(5) a large amount of distilled waters fully washed 4 minutes;
(6) collection tube is uncapped, put into 5 ℃ of clean refrigerators, leave standstill until collection tube lid and liquid in pipe and evaporate fully;
(7) take out collection tube, in super-clean bench, smear the poly-D-lysine that concentration is 4-8%, rapidly collection tube is put back to refrigerator, leave standstill 28h toward collection tube pipe interior surface stickiness module surface;
(8) repeating step (7) once.
Microdissection sample-collecting tubes cleans and utilizes method again, can implement successively as follows:
(1) in collection tube, adds the dimethylbenzene that accounts for collection tube volume 2/3, cover collection tube pipe lid, and vibrated 4 minutes, open the pipe lid, discard dimethylbenzene;
(2) in the described collection tube of step (1), add the suspension A that accounts for collection tube volume 1/2, the lid upper tube cap, and vibrated 3 minutes, open the pipe lid, the turned letter collection tube; Suspension A is mixed by hydrochloric acid, alcohol and EDTA, and adding 50mg diameter is made less than the silica dioxide granule of 0.1mm; Wherein the concentration of hydrochloric acid is 1.5%; The concentration of described alcohol is 30%; The concentration of described EDTA is 0.25mol/L; The volume ratio of hydrochloric acid, alcohol and EDTA is 0.5: 97: 2.
(3) in the described collection tube of step (2), add the distilled water that accounts for collection tube volume 1/2, add the silica dioxide granule of 50mg diameter simultaneously less than 0.1mm, the lid upper tube cap, and vibrated 2～3 minutes, open the pipe lid, the turned letter collection tube;
(4) repeating step (2) and step (3) twice successively;
(5) adopt distilled water that step (4) gained collection tube was washed 4 minutes;
(6) collection tube is uncapped, insert in 6 ℃ of refrigerators, evaporate fully until collection tube lid and liquid in pipe;
(7) take out collection tube, in super-clean bench, smear concentration toward collection tube pipe interior surface stickiness module surface and be 4～8% poly-D-lysine, rapidly collection tube is put back to refrigerator, leave standstill 48h;
(8) repeating step (7) once.
In order to verify that this cleaning utilizes the beneficial effect of method again, get two collection tubes, one is cleaned through this method, and one is cleaned without this method, carries out following experiment.In two arms, add the 80ul distilled water respectively, hatched 1 hour for 37 ℃.Take out 4ul liquid respectively as template, preparation 50ul standards system (dNTP:0.5ul, Taq enzyme: 0.5ul, 10nmol/ul upstream and downstream primer: each 0.5ul, 10 * buffer:5ul, ddH2O:43.5ul), carry out polymerase chain reaction,PCR (PCR), behind 40 cyclic amplifications, to get 6ul liquid respectively and put into 2% Ago-Gel and carry out electrophoresis such as Fig. 1, swimming lane 1 is the Marker sign; Swimming lane 2 is the sample after this method is handled, do not amplify any band, show that the collection tube after this method is cleaned has not had pollution of nucleic acid, this collection tube is after poly-D-lysine is handled, effectively keep and repaired collection tube pipe interior surface stickiness module adhesion, can continue on for micro-dissections; Swimming lane 3 is undressed sample, and nucleic acid-templated existence is obviously arranged.
Be with being appreciated that, more than about specific descriptions of the present invention, only be used to the present invention is described and be not to be subject to the described technical scheme of the embodiment of the invention, those of ordinary skill in the art is to be understood that, still can make amendment or be equal to replacement the present invention, to reach identical technique effect; Use needs as long as satisfy, all within protection scope of the present invention.
Claims (2)
1. microdissection sample-collecting tubes cleans and utilizes method again, it is characterized in that, implements successively as follows:
(1) in collection tube, adds the dimethylbenzene that accounts for collection tube volume 1/2～2/3, cover collection tube pipe lid, and vibrated 2～5 minutes, open the pipe lid, discard dimethylbenzene;
(2) in the described collection tube of step (1), add the suspension A that accounts for collection tube volume 1/2～2/3, the lid upper tube cap, and vibrated 2～3 minutes, open the pipe lid, the turned letter collection tube; Described suspension A is mixed by hydrochloric acid, alcohol and EDTA, and adding 50mg diameter is made less than the silica dioxide granule of 0.1mm; The concentration of described hydrochloric acid is 1.5%; The concentration of described alcohol is 30%; The concentration of described EDTA is 0.25mol/L; The volume ratio of described hydrochloric acid, alcohol and EDTA is 0.5～1.5: (96.5～99): 0.5～2;
(3) in the described collection tube of step (2), add the distilled water that accounts for collection tube volume 1/2～2/3, add the silica dioxide granule of 50mg diameter simultaneously less than 0.1mm, the lid upper tube cap, and vibrated 2～3 minutes, open the pipe lid, the turned letter collection tube;
(4) repeating step (2) and step (3) twice successively;
(5) adopt distilled water that step (4) gained collection tube was washed 3～5 minutes;
(6) collection tube is uncapped, insert in 2～8 ℃ of refrigerators, evaporate fully until collection tube lid and liquid in pipe;
(7) take out collection tube, in super-clean bench, smear concentration toward collection tube pipe interior surface stickiness module surface and be 4～8% poly-D-lysine, rapidly collection tube is put back to refrigerator, leave standstill;
(8) repeating step (7) once.
2. microdissection sample-collecting tubes according to claim 1 cleans and utilizes method again, it is characterized in that: time of repose t 〉=24h in the described step (7).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2009100121556A CN101587237B (en) | 2009-06-22 | 2009-06-22 | Method for cleaning and reusing microdissection sample-collecting tubes |
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| CN2009100121556A CN101587237B (en) | 2009-06-22 | 2009-06-22 | Method for cleaning and reusing microdissection sample-collecting tubes |
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| CN101587237A CN101587237A (en) | 2009-11-25 |
| CN101587237B true CN101587237B (en) | 2011-10-05 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6813008B2 (en) * | 2002-06-10 | 2004-11-02 | Palantyr Research, Llc | Microdissection optical system |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6813008B2 (en) * | 2002-06-10 | 2004-11-02 | Palantyr Research, Llc | Microdissection optical system |
Non-Patent Citations (1)
| Title |
|---|
| JP特开2005-15642A 2005.01.20 |
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