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Method for cleaning and reusing microdissection sample-collecting tubes

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Publication number
CN101587237B
CN101587237B CN 200910012155 CN200910012155A CN101587237B CN 101587237 B CN101587237 B CN 101587237B CN 200910012155 CN200910012155 CN 200910012155 CN 200910012155 A CN200910012155 A CN 200910012155A CN 101587237 B CN101587237 B CN 101587237B
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collecting
tube
adding
method
step
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CN 200910012155
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Chinese (zh)
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CN101587237A (en )
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陈洪铎
高兴华
齐瑞群
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中国医科大学附属第一医院
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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

显微切割标本收集管清洗再利用方法 Microdissection specimen collection tube cleaning method of recycling

技术领域 FIELD

[0001] 本发明涉及显微切割标本耗材清洗技术领域,具体的说是一种显微切割标本收集管清洗再利用方法。 [0001] The present invention relates to cleaning supplies microdissected specimens technical field, specifically a specimen collection tube cleaning microdissection method of recycling.

背景技术 Background technique

[0002] 激光捕获显微切割(Laser Capture Microdissection)技术及其系统的出现,使得相关科研工作者可以精确分离待研究的目的组织标本、细胞(体外培养的活细胞及固定在切片中组织细胞)、细胞器甚至染色体区带,用于后续研究,其控制样本的精确性受到全世界科研工作者的青睐,目前多数有实力的研究机构均已配备此技术及相关设施。 [0002] Laser capture microdissection (Laser Capture Microdissection) technology and its system occurs, so that researchers can be precisely related object isolated tissue sample to be investigated, cells (in vitro and in living cells in fixed tissue sections cells) , organelles and even chromosome zones for follow-up study, its accuracy control sample favored by the world's scientific researchers, for most powerful research institutions have been equipped with this technology and related facilities.

[0003] 用组织切片进行显微切割时,需要用到一种专用的耗材,该耗材为带盖的管形容器(以下简称收集管),其特征为收集管盖的内表面上携带表面光滑的特殊黏性模块,利用模块的黏性吸附力可收集显微切割时膜性载玻片上被激光切割分离的带有目标组织的膜。 [0003] When using microdissected tissue sections, the need to use a special consumables, the supplies for the tubular container lid (hereinafter referred to as collection tube), carried on the inner surface of the smooth surface wherein collection tube cap viscosity during a special module, with a module stickiness suction force may be collected by microdissection slides on the membrane film is cut with a separate laser target tissue. 这种收集管耗材价格非常昂贵;同时,由于用于研究的目的标本需要富集足够的数量,使得研究过程中该耗材使用数量较大,就单个标本而言,DNA、RNA相关的基因水平研究一般需要2-3个,蛋白水平的甚至需要数十个。 This collection tube supplies is very expensive; the same time, since the specimens for the purpose of study required enrichment sufficient quantity so that during the study used a large number of the consumable, in terms of on a single specimen, DNA, RNA gene level Correlation of it normally takes 2-3, protein levels even need dozens. 目前,国内仍没有制造商能够生产这种专用耗材,重复利用该耗材不失为一个好方法。 At present, the domestic manufacturers are still not able to produce this special supplies, re-use of the supplies is a good idea. 由于实验过程中激光会烧灼黏性模块表面致使出现焦痕, 从而降低其黏附性;同时,粘在盖子上的组织、核酸、蛋白及其他小分子物质会在后续分子生物学实验中造成污染。 Since the laser light is burning experiment sticky surface of the module so that scorching occurs, thereby reducing its adhesion; while, on the lid of the tissue adhesive, nucleic acids, proteins and other small molecules can cause pollution in the subsequent molecular biological experiments. 因此,如何能有效去除收集管管盖上的组织、核酸、蛋白及其他小分子物质,又不损伤收集管管盖特殊黏性模块的黏附性,且又能修复黏性模块表面在实验过程中被激光烧灼的焦痕,是重复利用该收集管的难题。 Therefore, how to effectively remove the collection tube covered with tissue, nucleic acids, proteins and other small molecules, does not damage the collection tube cap particular sticky adhesive property module and can fix the module surface stickiness during the experiment the laser burning scorch marks, the problem is to reuse collection tube. 常规机械洗涤、高温高压洗涤、强酸强碱洗涤等都会损伤收集管的黏附性,紫外线、钴60照射也只能灭活微生物,不能清除小分子物质。 Adhesion collection tube will damage a conventional washing machine, the high-temperature high-pressure washing, acid alkali washing, ultraviolet, cobalt 60 irradiation can inactivate microbes can not remove small molecules.

发明内容 SUMMARY

[0004] 本发明旨在克服现有技术的不足之处而提供一种能有效保持和修复收集管管盖内表面黏性模块黏附性,且能有效去除残留在收集管内的组织、核酸、蛋白及其他小分子物质,从而使收集管得以多次循环使用,节约大量实验经费的显微切割标本收集管清洗再利用方法。 [0004] The present invention aims to overcome the inadequacies of the prior art and to provide an effective repair and collection tube holder surface adhesion adhesive cover module, can effectively remove the residual tissue within the collection tube, nucleic acids, proteins and other small molecules, such that the collection tube to be reused many times, to save a large number of microscopic experimental expenditure cut specimen collection tube cleaning method of recycling.

[0005] 为达到上述目的,本发明是这样实现的: [0005] To achieve the above object, the present invention is implemented as follows:

[0006] 显微切割标本收集管清洗再利用方法,可按如下步骤依次实施: [0006] microdissected specimen collection tube cleaning method of recycling, the steps may be implemented sequentially:

[0007] (1)向收集管中加入占收集管容积1/2〜2/3的二甲苯,盖上收集管管盖,并振荡2〜5分钟,打开管盖,弃去二甲苯; [0007] (1) was added to the collection tube to collect accounting xylene pipe volume 1 / 2~2 / 3, collection tube covered with the cover, and shaken 2 ~ 5 minutes, the tube was opened cover discarded xylene;

[0008] (2)向步骤(1)所述收集管中加入占收集管容积1/2〜2/3的混悬液A,盖上管盖, 并振荡2〜3分钟,打开管盖,倒空收集管; [0008] (2) of step (1) was added to the collection tube collection tubes volume accounts for 1 / 2~2 / 3 suspension A, Cap the tube and shaken for 2 to 3 minutes, the tube was opened cover, empty the collection tube;

[0009] (3)向步骤(¾所述收集管中加入占收集管容积1/2〜2/3的双蒸水,同时加入50mg直径小于0. Imm的二氧化硅颗粒,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管; [0009] (3) the step of (¾ representing the collection tube was added distilled water collecting pipe volume 1 / 2~2 / 3, while the addition of the silica particles diameter of less than 0. Imm of 50mg, Cap the tube , and shaken for 2 to 3 minutes, the tube was opened cover, emptied collection tube;

[0010] (4)依次重复步骤⑵及步骤⑶两次; [0010] (4) and the step of sequentially repeating the step ⑵ ⑶ twice;

[0011] (5)采用双蒸水对步骤(4)所得收集管进行冲洗3〜5分钟; [0011] (5) using double distilled water in step (4) was collected by rinsing tube 3 ~ 5 min;

[0012] (6)将收集管开盖,置入2〜8°C冰箱内,直至收集管盖及管内液体完全蒸发; [0012] (6) The collector tube open end, into the inner 2~8 ° C freezer until the liquid collection tube cap, and the tube was completely evaporated;

[0013] (7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4〜 8%的多聚赖氨酸,迅速将收集管放回冰箱,静置; [0013] (7) remove the collection tube in a clean bench, to collect the applicator surface concentration in the tube surface of the module cover viscosity of 4 ~ 8% polylysine, quickly collection tube back into the refrigerator, static set;

[0014] (8)重复步骤(7) —次。 [0014] (8) repeating steps (7) - times.

[0015] 作为一种优选方案,本发明所述混悬液A由盐酸、酒精及EDTA混配,并加入50mg 直径小于0. Imm的二氧化硅颗粒制成。 [0015] As a preferred embodiment, the present invention A suspension was mixed with hydrochloric acid, alcohol and EDTA, and added to the silica particle diameter of less than 0. Imm 50mg made.

[0016] 作为另一种优选方案,本发明所述盐酸的浓度为1. 5% ;所述酒精的浓度为30% ; 所述EDTA的浓度为0. 25mol/L。 [0016] As another preferred embodiment, the concentration of hydrochloric acid of the present invention was 1.5%; the alcohol concentration is 30%; the concentration of EDTA is 0. 25mol / L.

[0017] 进一步地,本发明所述盐酸、酒精及EDTA的体积比为0.5〜1.5 : (96. 5〜 99) : 0. 5 〜2。 [0017] Further, according to the present invention, the volume ratio of the hydrochloric acid, alcohol and EDTA is 0.5~1.5: (-5 to 96. 99): 0.5 ~ 2.

[0018] 更进一步地,本发明所述步骤(7)中静置时间t彡24h。 [0018] Still further, the present invention, the step (7) resting time t San 24h.

[0019] 本发明与现有技术相比具有如下特点: [0019] The present invention as compared with the prior art having the following characteristics:

[0020] 1、本发明整个操作流程中不涉及损伤暴力、高温高压、强酸碱等对膜有损害的因素; [0020] 1, do not involve violent damage factors, high temperature and pressure, strong acid and other damage to the film flow of the overall operation of the present invention;

[0021] 2、本发明通过使用混悬液A,使黏性模块得到充分清洗,同时充分保护了模块的黏附性; [0021] 2, by using the present invention, a suspension A, so that the viscous module are fully washed, while fully protecting the adhesion module;

[0022] 3、本发明通过往表面涂抹多聚赖氨酸,可以很好的修复激光在黏性模块表面造成的烧痕,并在一定程度上恢复烧痕部位的黏附性; [0022] 3, by the present invention is applied to the surface of the poly-lysine, well repair burn marks due to the viscosity of the laser module surface, and to restore a certain degree burn marks on portions of the adhesion;

[0023] 4、本发明清洗后的收集管不含有对实验有影响的成分; [0023] 4, after the collection tube of the present invention the cleaning composition does not contain an impact on experimental;

[0024] 5、本发明所需试剂均为生物实验室常规试剂,且价格低廉,可批量操作; [0024] 5, the present invention is desired biological laboratory reagents are conventional reagents, and inexpensive, may be a batch operation;

[0025] 6、本发明重复利用率高,可节省10-20倍的收集管数量,节省大量科研经费。 [0025] 6, the present invention is a high recycle rate, can save 10 to 20 times the number of collection tube, save a lot of research funding.

附图说明 BRIEF DESCRIPTION

[0026] 下面结合附图和具体实施方式对本发明作进一步说明。 [0026] The present invention will be further described in conjunction with the accompanying drawings and specific embodiments. 本发明的保护范围不仅局限于下列内容的表述。 The scope of the present invention is not limited to the following expression.

[0027] 图1为验证收集管是否清洗干净而进行的2%的琼脂糖电泳图。 [0027] Figure 1 is collected to verify if the cleaning tube 2% agarose gel electrophoresis FIG clean carried out.

具体实施方式 detailed description

[0028] 实施例1 [0028] Example 1

[0029] 显微切割标本收集管清洗再利用方法,可按如下步骤依次实施: [0029] microdissected specimen collection tube cleaning method of recycling, the steps may be implemented sequentially:

[0030] (1)向收集管中加入占收集管容积1/2的二甲苯,盖上管盖,涡旋器振荡3分钟,打 [0030] (1) was added to the collection tube and collection tube volume accounts for 1/2 xylene, Cap the tube vortexer shaken for 3 minutes, playing

开管盖,弃去二甲苯; Lid opening tube, xylene was discarded;

[0031] (2)向收集管中加入占收集管容积2/3的混悬液A(由1.5%盐酸、30%酒精、 0. 25mol/L EDTA按体积比1 : 97 : 1混合,并加入50mg直径小于0. Imm的二氧化硅颗粒配制而成)。 [0031] (2) was added to the collection tube and 2/3 volume of accounting collection tube suspension A (from 1.5% hydrochloric acid, 30% ethanol, 0. 25mol / L EDTA volume ratio of 1: 97: 1 mixture, and was added 50mg diameter of less than 0. Imm preparation of silica particles). 盖上管盖,涡旋器振荡2. 5分钟,打开管盖,倒空收集管; Cap the tube, vortex oscillation 2.5 minutes, the tube was opened cover, emptied collection tube;

[0032] (3)向收集管中加入占收集管容积1/2的双蒸水,同时加入50mg直径小于0. Imm [0032] (3) the volume of the collection tube was added accounted for 1/2 of double distilled water, and added to 50mg diameter of less than 0. Imm collection tube

4的二氧化硅颗粒,盖上管盖,涡旋器振荡2. 5分钟,打开管盖,倒空收集管; Silica particles 4, Cap the tube, vortex oscillation 2.5 minutes, the tube was opened cover, emptied collection tube;

[0033] (4)重复步骤(2)及步骤(3)两次; [0033] (4) repeating steps (2) and the step (3) twice;

[0034] (5)大量双蒸水充分冲洗4分钟; [0034] (5) sufficiently large number of double distilled water rinse 4 minutes;

[0035] (6)将收集管开盖,放入洁净的5°C冰箱,静置直至收集管盖及管内液体完全蒸发; [0035] (6) The collector tube open end, into a clean 5 ° C of a refrigerator, and left to stand until the liquid collection tube cap inner tube complete evaporation;

[0036] (7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4-8%的多聚赖氨酸,迅速将收集管放回冰箱,静置^h ; [0036] (7) remove the collection tube in a clean bench, to collect the applicator surface concentration in the tube surface of the module cover viscosity of 4-8% polylysine, quickly collection tube back into the refrigerator, static set ^ h;

[0037] (8)重复步骤(7) —次。 [0037] (8) repeating steps (7) - times.

[0038] 实施例2 [0038] Example 2

[0039] 显微切割标本收集管清洗再利用方法,可按如下步骤依次实施: [0039] microdissected specimen collection tube cleaning method of recycling, the steps may be implemented sequentially:

[0040] (1)向收集管中加入占收集管容积2/3的二甲苯,盖上收集管管盖,并振荡4分钟, 打开管盖,弃去二甲苯; [0040] (1) was added to the collection tube and collection tube volume accounted for 2/3 of the xylene, the collection tube covered with the cover and shaken for 4 minutes to open the cover tube, xylene was discarded;

[0041] (2)向步骤(1)所述收集管中加入占收集管容积1/2的混悬液A,盖上管盖,并振荡3分钟,打开管盖,倒空收集管;混悬液A由盐酸、酒精及EDTA混配,并加入50mg直径小于0. Imm的二氧化硅颗粒制成;其中盐酸的浓度为1. 5% ;所述酒精的浓度为30% ;所述EDTA 的浓度为0. 25mol/L;盐酸、酒精及EDTA的体积比为0. 5 : 97 : 2。 [0041] (2) of step (1) of the collection tube were added 1/2 volume accounted suspension collection tube A, Cap the tube and shaken for 3 minutes, the tube was opened cover, emptied collection tube; mix a suspension was mixed with hydrochloric acid, alcohol and EDTA, and added to the silica particle diameter of less than 0. Imm of 50mg formed; wherein the concentration of hydrochloric acid was 1.5%; the alcohol concentration of 30%; the EDTA concentration of 0. 25mol / L; hydrochloric acid volume ratio of alcohol and EDTA is 0.5: 97: 2.

[0042] (3)向步骤(2)所述收集管中加入占收集管容积1/2的双蒸水,同时加入50mg直径小于0. Imm的二氧化硅颗粒,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管; [0042] (3) step (2) was added to the collection tube collection tubes occupy 1/2 volume double-distilled water, while adding silica particles diameter of less than 0. Imm of 50mg, Cap the tube and shaken 2 or 3 minutes, the tube was opened cover, emptied collection tube;

[0043] (4)依次重复步骤(2)及步骤(3)两次; [0043] (4) sequentially repeating steps (2) and the step (3) twice;

[0044] (5)采用双蒸水对步骤(4)所得收集管进行冲洗4分钟; [0044] (5) using double distilled water in step (4) was collected by rinsing the tube 4 min;

[0045] (6)将收集管开盖,置入6°C冰箱内,直至收集管盖及管内液体完全蒸发; [0045] (6) The collector tube opening the lid placed within 6 ° C freezer until the liquid collection tube cap, and the tube was completely evaporated;

[0046] (7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4〜 8%的多聚赖氨酸,迅速将收集管放回冰箱,静置48h ; [0046] (7) remove the collection tube in a clean bench, to collect the applicator surface concentration in the tube surface of the module cover viscosity of 4 ~ 8% polylysine, quickly collection tube back into the refrigerator, static set 48h;

[0047] (8)重复步骤(7) —次。 [0047] (8) repeating steps (7) - times.

[0048] 为了验证本清洗再利用方法的有益效果,取两支收集管,一支经过本方法清洗,一支未经本方法清洗,进行以下实验。 [0048] In order to verify the present cleaning method of recycling a beneficial effect, take two collection tube, through a cleaning method of the present, the present method without a cleaning, the following experiment. 分别向两支管中加入80ul双蒸水,37°C孵育1小时。 80ul distilled water were added to two tubes, 37 ° C for 1 hour. 分别取出4ul液体作为模板,配制50ul标准体系(dNTP :0. 5ul,Taq酶:0. 5ul,10nmol/ul上下游引物:各0. 5ul,IOXbuffer :5ul,ddH20 :43. 5ul),进行多聚酶链反应(PCR),经40个循环扩增后,分别取6ul液体放入2%琼脂糖凝胶进行电泳如图1,泳道1为Marker标志; 泳道2为经本方法处理后的样本,未扩增出任何条带,表明经本方法清洗后的收集管已经没有核酸污染,该收集管经多聚赖氨酸处理后,有效保持和修复了收集管管盖内表面黏性模块黏附性,可以继续用于显微切割;泳道3为未经处理的样本,明显有核酸模板存在。 Are taken 4ul liquid as a template to prepare 50ul standards (dNTP:. 0 5ul, Taq enzyme:. 0 5ul, 10nmol / upper ul downstream primers: 0. 5ul, IOXbuffer: 5ul, ddH20:. 43 5ul), for polymerase chain reaction (the PCR), after 40 cycles of amplification were taken 6ul liquid into a 2% agarose gel electrophoresis in FIG. 1, lane 1 is a marker marker; lane 2 is a sample treated by the present process, not any amplified bands revealed that a collection tube after cleaning of the present method has no nucleic acid contamination, after the collection tube via polylysine treatment, effective to maintain and repair the inner surface of the collection tube cap viscous adhesion module, It may continue to be used microdissection; lane 3 untreated sample, obviously nucleic acid template present.

[0049] 可以理解地是,以上关于本发明的具体描述,仅用于说明本发明而并非受限于本发明实施例所描述的技术方案,本领域的普通技术人员应当理解,仍然可以对本发明进行修改或等同替换,以达到相同的技术效果;只要满足使用需要,都在本发明的保护范围之内。 [0049] be understood that the foregoing detailed description of the present invention, for merely illustrative and the present invention is not limited to the technical solutions described in the embodiments of the present invention, those of ordinary skill in the art will be appreciated that the present invention may still modifications or equivalents, to achieve the same technical effect; use as long as needed, are within the scope of the present invention.

5 5

Claims (2)

1.显微切割标本收集管清洗再利用方法,其特征在于,按如下步骤依次实施:(1)向收集管中加入占收集管容积1/2〜2/3的二甲苯,盖上收集管管盖,并振荡2〜 5分钟,打开管盖,弃去二甲苯;(2)向步骤(1)所述收集管中加入占收集管容积1/2〜2/3的混悬液A,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管;所述混悬液A由盐酸、酒精及EDTA混配,并加入50mg 直径小于0. Imm的二氧化硅颗粒制成;所述盐酸的浓度为1. 5% ;所述酒精的浓度为30% ; 所述EDTA的浓度为0. 25mol/L ;所述盐酸、酒精及EDTA的体积比为0. 5〜1. 5 : (96. 5〜 99) : 0. 5 〜2 ;(3)向步骤(2)所述收集管中加入占收集管容积〜2/3的双蒸水,同时加入50mg 直径小于0. Imm的二氧化硅颗粒,盖上管盖,并振荡2〜3分钟,打开管盖,倒空收集管;(4)依次重复步骤(¾及步骤(¾两次;(5)采用双蒸水对步骤(4)所 1. microdissected specimen collection tube cleaning method of recycling, characterized in that, in sequence, the following embodiments: (1) collection tube was added to the xylene accounted for collecting pipe volume 1 / 2~2 / 3, collection tube cap tube cap, and shaken for 2 ~ 5 minutes, the tube was opened cover discarded xylene; (2) of step (1) was added to the collection tube to collect accounting pipe volume suspension a 1 / 2~2 / 3, and cap the tube and shaken for 2 to 3 minutes, the tube was opened cover, emptied collection tube; the suspension was mixed with hydrochloric acid a, alcohol and EDTA, and added 50mg diameter of less than 0. Imm silica particles prepared to; the concentration of hydrochloric acid was 1.5%; the alcohol concentration is 30%; the concentration of EDTA is 0. 25mol / L; hydrochloric acid, the volume ratio of alcohol and EDTA is 0. 5~1 . 5: (-5 to 96. 99): 0.5 ~ 2; (3) step (2) accounts for the collection tube was added distilled water collecting pipe volume ~ 2/3, while a smaller diameter than 0 was added 50mg . Imm silica particles, cap the tube and shaken for 2 to 3 minutes, the tube was opened cover, emptied collection tube; (4) sequentially repeating steps (¾ and step (¾ twice; (5) double distilled water in step (4) 得收集管进行冲洗3〜5分钟;(6)将收集管开盖,置入2〜8°C冰箱内,直至收集管盖及管内液体完全蒸发;(7)取出收集管,在超净台内,往收集管管盖内表面黏性模块表面涂抹浓度为4〜8% 的多聚赖氨酸,迅速将收集管放回冰箱,静置;(8)重复步骤(7) 一次。 Rinsed 3 to 5 have the collection tube min; (6) the collection tube open end, into the inner 2~8 ° C freezer until the liquid collection tube cap, and the tube was completely evaporated; (7) remove the collection tube, in Clean Bench within, the inner surface of the lid to the collection tube adhesive applicator module 4 to 8% of the surface concentration of polylysine, quickly collection tube back into the refrigerator, standing; (8) repeating steps (7) again.
2.根据权利要求1所述的显微切割标本收集管清洗再利用方法,其特征在于:所述步骤(7)中静置时间t彡24h。 2. The microstructure of claim 1, cutting the specimen collection tube cleaning method of recycling, characterized in that: said step (7) of the standing time t San 24h.
CN 200910012155 2009-06-22 2009-06-22 Method for cleaning and reusing microdissection sample-collecting tubes CN101587237B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6813008B2 (en) 2002-06-10 2004-11-02 Palantyr Research, Llc Microdissection optical system

Patent Citations (1)

* Cited by examiner, † Cited by third party
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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* Cited by examiner, † Cited by third party
Title
JP特开2005-15642A 2005.01.20

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