CN109212006A - A kind of unicellular agarose gel electrophoresis kit - Google Patents
A kind of unicellular agarose gel electrophoresis kit Download PDFInfo
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- CN109212006A CN109212006A CN201810970225.8A CN201810970225A CN109212006A CN 109212006 A CN109212006 A CN 109212006A CN 201810970225 A CN201810970225 A CN 201810970225A CN 109212006 A CN109212006 A CN 109212006A
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- China
- Prior art keywords
- silver staining
- comet
- staining reagent
- electrophoresis
- unicellular
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
Abstract
The present invention relates to field of biotechnology, are concretely related to a kind of unicellular agarose gel electrophoresis kit, consist of the following reagents: 40~50ml of electrophoresis reagents, 40~60ml of silver staining reagent and 80~130ul of secure attachment agent；The electrophoresis reagents include 30~56ml of LS lysate, 2~10ml of LMA agar, TC comet electrophoresis specialized glass slide and 0.5~2mM EDTA；Using electrophoresis kit of the invention in comet, it can be observed that the fragment (tail of a comet) migrated from core (comet head)；Negatively charged DNA is migrated to anode, squeeze out the increase relaxation that length reflects supercoil, the common description of DNA damage is the DNA percentage on tail and tail of a comet moment in Comet Assay, and the DNA percentage on the tail of a comet is the standardization measurement to total cell dna percentage on the tail of a comet；This kind of kit can be readily generated quantitative and statistical data, with figuring the tail length, the time of tail portion DNA and tail, scientific research is suitble to promote the use of.
Description
Technical field
The present invention relates to field of biotechnology, are concretely related to a kind of unicellular agarose gel electrophoresis kit.
Background technique
Trevigen'sOr single cell gel electrophoresis test, provide a kind of simple and effective method
To assess in DNA Damage.The principle of this detection is based under the influence of electric fields, and the denaturation of DNA, crack fragment can
It is migrated out from nucleotide, and undamaged DNA migration velocity is slower, and remains at nucleotide when applying electric current
In range.DNA damage can be assessed to the assessment of DNA " comet " tail shape and migration model.It is neutralUsually
For detecting double-strand break, and it is alkalineIt is more sensitive, and include that single, double chain is disconnected for detecting a small amount of injury
It splits.
Trevigen'sIt is special using our exclusive CometSlideTMProcessing is to promote low melting point fine jade
The adherency of lipolysaccharide.This eliminates time-consuming and insecure conventional method, prepares the agarose of base.Use Trevigen's
CometSlideTMShorten test period, and allows the quickly analysis with reliable a large amount of samples.Trevigen'sThe Silver stain for all reagents that silver-colored kit provides processes CometSlideTMAllow to visualize normalized optical aobvious
Micro mirror and offer permanent stain sample filing.
It is chemically examined in comet, cell is fixed low melting-point agarose in bed, in Trevigen CometSlideTM.It is gentle
Cell cracking after, alkalinityThe DNA that sample treatment alkali loosens and is denaturalized is damaged with hydrolysis website.To both
For method, cell is all dissolved, and remaining nucleic is by electrophoresis and subsequent dyeing and fluorescent DNA insertion dyestuff and/or silver
Dyeing.
Trevigen suggests using alkalinityCell is controlled when the alkaline electrophoresis of execution and neutralityControl reproducibility of the cell when the neutral Comet Assay of execution, between monitoring test condition and verifying independent operating.Gold DNA visualization and quantitative microscopic digital suggestion.Silver stain can replace or track fluorescence analysis.
It is proposed that using Trevigen'sElectrophoresis system is intended to eliminate the reason of known analysis variation.
Electrophoresis step is alkaline version to be carried out using alkaline electrophoresis pH value of solution > 13, and neutral electrophoresis then recommends neutral electrophoretic buffer.It is logical
It crosses and fluorescence analysis is carried out to result using the Comet analysis software of Trevigen, quantitative and statistical number can be readily generated
According to figuring the tail length, the time of tail portion DNA and tail.
Summary of the invention
Therefore the present invention proposes a kind of unicellular agarose gel electrophoresis kit, for solving asking for above-mentioned background technique
Topic.
The technical scheme of the present invention is realized as follows: a kind of unicellular agarose gel electrophoresis kit, by following examination
Agent composition: 40~50ml of electrophoresis reagents, 40~60ml of silver staining reagent and 80~130ul of secure attachment agent.
Further, the electrophoresis reagents include that 30~56ml of LS lysate, 2~10ml of LMA agar, TC comet electrophoresis are special
With glass slide and 0.5~2mM EDTA.
Further, it consists of the following reagents: 40~50ml of electrophoresis reagents, 40~50ml of silver staining reagent, secure attachment agent
80~130ul；The electrophoresis reagents include 36~50ml of LS lysate, 2~10ml of LMA agar, the dedicated load glass of TC comet electrophoresis
Piece and 0.5~2mM EDTA.
Further, the LS lysate is made of following ingredients: 150mM sodium chloride, 1.0%NP-40 (detergent),
0.1%SDS (detergent), 2.0ug/ml, Aprotinin (protease inhibitors), 2.0ug/ml Leupeptin (protease suppression
Preparation), 1mM PMSF (protease inhibitors), 1.5mM EDTA (protease inhibitors), 1.0mM Na Vanadate (phosphoric acid
Esterase inhibitor), the mentioned reagent is dissolved in proportion in 150mM sodium chloride solution.
Further, the LMA agar is 0.5% agarose of concentration.
Further, the secure attachment agent includes 5~20ul of fixative, 10~50ul of distilled water, 30~80ul of methanol
With 5~20ul of glacial acetic acid.
Further, the fixative is slow including 2.0% paraformaldehyde 25ml, 5% glutaraldehyde 5.0ml, 0.2M phosphate
Fliud flushing 20ml, 0.025g calcium chloride.
Further, the silver staining reagent includes silver staining reagent 1, silver staining reagent 2, silver staining reagent 3, silver staining reagent 4；It is described
Silver staining reagent 1 forms with 24% ethyl alcohol and 0.6% benzene sulfonic acid fixer；The silver staining reagent 2 by 0.2% silver nitrate and
0.007% benzene sulfonic acid dyeing liquor mixes；The silver staining reagent 3 is thio by 2.5% sodium carbonate, 0.1% formaldehyde and 0.002%
Sodium sulphate developer solution is prepared；The silver staining reagent 4 is by 1.0% glacial acetic acid, 5.0% sodium acetate and 10% glycerine terminate liquid
Cooperate.
By above disclosure, the invention has the benefit that using electrophoresis kit of the invention in comet
In, in the cell for having had accumulated DNA damage, it can be observed that the fragment (tail of a comet) migrated from core (comet head)；Band is negative
The DNA of charge is migrated to anode, squeezes out the increase relaxation that length reflects supercoil, the common of DNA damage is retouched in Comet Assay
Stating is DNA percentage and tail of a comet moment on tail, and the DNA percentage on the tail of a comet is to total cell dna percentage on the tail of a comet
Standardization measurement；This kind of kit can be readily generated quantitative and statistical data, with figuring the tail length, tail portion DNA and
The time of tail is suitble to scientific research to promote the use of.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Based on of the invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The present invention proposes a kind of unicellular agarose gel electrophoresis kit.
A kind of unicellular agarose gel electrophoresis kit, consists of the following reagents: 40~50ml of electrophoresis reagents, silver staining examination
80~130ul of 40~60ml of agent and secure attachment agent；Electrophoresis reagents include 30~56ml of LS lysate, 2~10ml of LMA agar,
TC comet electrophoresis specialized glass slide and 0.5~2mM EDTA；LS lysate is made of following ingredients: 150mM sodium chloride, 1.0%
NP-40 (detergent), 0.1%SDS (detergent), 2.0ug/ml, Aprotinin (protease inhibitors), 2.0ug/ml
Leupeptin (protease inhibitors), 1mM PMSF (protease inhibitors), 1.5mM EDTA (protease inhibitors), 1.0mM
Na Vanadate (phosphatase inhibitors), mentioned reagent are dissolved in proportion in 150mM sodium chloride solution；LMA agar is dense
Spend 0.5% agarose；The secure attachment agent includes 5~20ul of fixative, 10~50ul of distilled water, 30~80ul of methanol and ice
5~20ul of acetic acid；Fixative include 2.0% paraformaldehyde 25ml, 5% glutaraldehyde 5.0ml, 0.2M phosphate buffer 20ml,
0.025g calcium chloride.
Silver staining reagent includes silver staining reagent 1, silver staining reagent 2, silver staining reagent 3, silver staining reagent 4；Silver staining reagent 1 is by 24% second
Pure and mild 0.6% benzene sulfonic acid fixer cooperates；Silver staining reagent 2 is mixed by 0.2% silver nitrate and 0.007% benzene sulfonic acid dyeing liquor
It forms；Silver staining reagent 3 is prepared by 2.5% sodium carbonate, 0.1% formaldehyde and 0.002% sodium thiosulfate developer solution；Silver staining examination
Agent 4 forms with 1.0% glacial acetic acid, 5.0% sodium acetate and 10% glycerine terminate liquid.
Embodiment 1
A kind of unicellular agarose gel electrophoresis kit, consists of the following reagents:
Electrophoresis reagents 40ml, electrophoresis reagents include LS lysate, LMA agar, TC comet electrophoresis specialized glass slide, EDTA；
LMA agar is 0.6% agarose；The amount of EDTA is 1.0mM；LS lysate is made of following ingredients: 150mM sodium chloride, 1.0%
NP-40 (detergent), 0.1%SDS (detergent), 2.0ug/ml, Aprotinin (protease inhibitors), 2.0ug/ml
Leupeptin (protease inhibitors), 1mM PMSF (protease inhibitors), 1.5mM EDTA (protease inhibitors), 1.0mM
Na Vanadate (phosphatase inhibitors), mentioned reagent are dissolved in proportion in 150mM sodium chloride solution.
Silver staining reagent 40ml, silver staining reagent include 10ml silver staining reagent 1,10ml silver staining reagent 2,10ml silver staining reagent 3,
10ml silver staining reagent 4；Silver staining reagent 1 forms with 22% ethyl alcohol and 0.8% benzene sulfonic acid fixer；Silver staining reagent 2 is by 0.1%
Silver nitrate and 0.008% benzene sulfonic acid dyeing liquor mix；Silver staining reagent 3 is by 2.2% sodium carbonate, 0.1% formaldehyde and 0.002%
Sodium thiosulfate developer solution is prepared；Silver staining reagent 4 is by 1.5% glacial acetic acid, 5.0% sodium acetate and 20% glycerine terminate liquid
Cooperate.
Secure attachment agent 80ul, secure attachment agent include fixative 5ul, 10~50ul of distilled water, 30~80ul of methanol and
After glacial acetic acid 5~20ul mixed preparing, 80ul mixed liquor is taken.(it is to be noted that fixative includes 2.0% paraformaldehyde
5ul is taken after 25ml, the mixing of 5% glutaraldehyde 5.0ml, 0.2M phosphate buffer 20ml, 0.025g calcium chloride)
Embodiment 2
A kind of unicellular agarose gel electrophoresis kit, consists of the following reagents:
Electrophoresis reagents 45ml, electrophoresis reagents include LS lysate, LMA agar, TC comet electrophoresis specialized glass slide, EDTA；
LMA agar is 0.5% agarose；The amount of EDTA is 1.5mM；LS lysate is made of following ingredients: 150mM sodium chloride, 1.0%
NP-40 (detergent), 0.1%SDS (detergent), 2.0ug/ml, Aprotinin (protease inhibitors), 2.0ug/ml
Leupeptin (protease inhibitors), 1mM PMSF (protease inhibitors), 1.5mM EDTA (protease inhibitors), 1.0mM
Na Vanadate (phosphatase inhibitors), mentioned reagent are dissolved in proportion in 150mM sodium chloride solution.
Silver staining reagent 60ml, silver staining reagent include 15ml silver staining reagent 1,15ml silver staining reagent 2,15ml silver staining reagent 3,
15ml silver staining reagent 4；Silver staining reagent 1 forms with 24% ethyl alcohol and 0.6% benzene sulfonic acid fixer；Silver staining reagent 2 is by 0.2%
Silver nitrate and 0.007% benzene sulfonic acid dyeing liquor mix；Silver staining reagent 3 is by 2.5% sodium carbonate, 0.1% formaldehyde and 0.002%
Sodium thiosulfate developer solution is prepared；Silver staining reagent 4 is by 1.0% glacial acetic acid, 5.0% sodium acetate and 10% glycerine terminate liquid
Cooperate.
Secure attachment agent 90ul, secure attachment agent include fixative 8ul, 10~50ul of distilled water, 30~80ul of methanol and
After glacial acetic acid 5~20ul mixed preparing, 90ul mixed liquor is taken.(it is to be noted that fixative includes 2.0% paraformaldehyde
8ul is taken after 25ml, the mixing of 5% glutaraldehyde 5.0ml, 0.2M phosphate buffer 20ml, 0.025g calcium chloride)
Embodiment 3
A kind of unicellular agarose gel electrophoresis kit, consists of the following reagents:
Electrophoresis reagents 50ml, electrophoresis reagents include LS lysate, LMA agar, TC comet electrophoresis specialized glass slide, EDTA；
LMA agar is 0.8% agarose；The amount of EDTA is 1.0mM；LS lysate is made of following ingredients: 150mM sodium chloride, 1.0%
NP-40 (detergent), 0.1%SDS (detergent), 2.2ug/ml, Aprotinin (protease inhibitors), 2.5ug/ml
Leupeptin (protease inhibitors), 1mM PMSF (protease inhibitors), 1.5mM EDTA (protease inhibitors), 1.5mM
Na Vanadate (phosphatase inhibitors), mentioned reagent are dissolved in proportion in 200mM sodium chloride solution.
Silver staining reagent 50ml, silver staining reagent include 12.5ml silver staining reagent 1,12.5ml silver staining reagent 2, the examination of 12.5ml silver staining
Agent 3,12.5ml silver staining reagent 4；Silver staining reagent 1 forms with 30% ethyl alcohol and 0.8% benzene sulfonic acid fixer；Silver staining reagent 2
It is mixed by 0.3% silver nitrate and 0.010% benzene sulfonic acid dyeing liquor；Silver staining reagent 3 by 2.5% sodium carbonate, 0.1% formaldehyde and
0.002% sodium thiosulfate developer solution is prepared；Silver staining reagent 4 is by 1.0% glacial acetic acid, 5.0% sodium acetate and 10% the third three
Alcohol terminate liquid cooperates.
Secure attachment agent 100ul, secure attachment agent include fixative 10ul, 10~50ul of distilled water, 30~80ul of methanol
After glacial acetic acid 5~20ul mixed preparing, 100ul mixed liquor is taken.(it is to be noted that fixative includes 2.0% poly
10ul is taken after formaldehyde 25ml, the mixing of 5% glutaraldehyde 5.0ml, 0.2M phosphate buffer 20ml, 0.025g calcium chloride)
Embodiment 4
A kind of unicellular agarose gel electrophoresis kit, consists of the following reagents:
Electrophoresis reagents 50ml, electrophoresis reagents include LS lysate, LMA agar, TC comet electrophoresis specialized glass slide, EDTA；
LMA agar is 0.8% agarose；The amount of EDTA is 1.0mM；LS lysate is made of following ingredients: 150mM sodium chloride, 1.0%
NP-40 (detergent), 0.1%SDS (detergent), 2.2ug/ml, Aprotinin (protease inhibitors), 2.5ug/ml
Leupeptin (protease inhibitors), 1mM PMSF (protease inhibitors), 1.5mM EDTA (protease inhibitors), 1.5mM
Na Vanadate (phosphatase inhibitors), mentioned reagent are dissolved in proportion in 200mM sodium chloride solution.
Silver staining reagent 60ml, silver staining reagent include 12.5ml silver staining reagent 1,12.5ml silver staining reagent 2, the examination of 12.5ml silver staining
Agent 3,12.5ml silver staining reagent 4；Silver staining reagent 1 forms with 30% ethyl alcohol and 0.8% benzene sulfonic acid fixer；Silver staining reagent 2
It is mixed by 0.3% silver nitrate and 0.010% benzene sulfonic acid dyeing liquor；Silver staining reagent 3 by 2.5% sodium carbonate, 0.1% formaldehyde and
0.002% sodium thiosulfate developer solution is prepared；Silver staining reagent 4 is by 1.0% glacial acetic acid, 5.0% sodium acetate and 10% the third three
Alcohol terminate liquid cooperates.
Secure attachment agent 120ul, secure attachment agent include fixative 20ul, 10~50ul of distilled water, 30~80ul of methanol
After glacial acetic acid 5~20ul mixed preparing, 100ul mixed liquor is taken.(it is to be noted that fixative includes 2.0% poly
20ul is taken after formaldehyde 25ml, the mixing of 5% glutaraldehyde 5.0ml, 0.2M phosphate buffer 20ml, 0.025g calcium chloride)
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (8)
1. a kind of unicellular agarose gel electrophoresis kit, it is characterised in that: consist of the following reagents: electrophoresis reagents 40~
50ml, 40~60ml of silver staining reagent and 80~130ul of secure attachment agent.
2. a kind of unicellular agarose gel electrophoresis kit according to claim 1, it is characterised in that: the electrophoresis examination
Agent includes 30~56ml of LS lysate, 2~10ml of LMA agar, TC comet electrophoresis specialized glass slide and 0.5~2mM EDTA.
3. a kind of unicellular agarose gel electrophoresis kit according to claim 2, it is characterised in that: by following reagent
Composition: 40~50ml of electrophoresis reagents, 40~50ml of silver staining reagent and 86~110ul of secure attachment agent；The electrophoresis reagents include
36~50ml of LS lysate, 2~10ml of LMA agar, TC comet electrophoresis specialized glass slide and 0.5~2mM EDTA.
4. a kind of unicellular agarose gel electrophoresis kit according to claim 2, it is characterised in that: the LS cracking
Liquid is made of following ingredients: 150mM sodium chloride, 1.0%NP-40,0.1%SDS, 2.0ug/ml, Aprotinin, 2.0ug/ml
Leupeptin, 1mM PMSF, 1.5mM EDTA, 1.0mM Na Vanadate, the mentioned reagent are dissolved in 150mM in proportion
In sodium chloride solution.
5. a kind of unicellular agarose gel electrophoresis kit according to claim 2, it is characterised in that: the LMA fine jade
Rouge is 0.5% agarose of concentration.
6. a kind of unicellular agarose gel electrophoresis kit according to claim 1, it is characterised in that: the fixation is viscous
Attached dose includes 5~20ul of fixative, 5~20ul of 10~50ul of distilled water, 30~80ul of methanol and glacial acetic acid.
7. a kind of unicellular agarose gel electrophoresis kit according to claim 6, it is characterised in that: the fixative
Including 2.0% paraformaldehyde 25ml, 5% glutaraldehyde 5.0ml, 0.2M phosphate buffer 20ml, 0.025g calcium chloride.
8. a kind of unicellular agarose gel electrophoresis kit according to claim 7, it is characterised in that: the silver staining examination
Agent includes silver staining reagent 1, silver staining reagent 2, silver staining reagent 3, silver staining reagent 4；The silver staining reagent 1 is by 24% ethyl alcohol and 0.6%
Benzene sulfonic acid fixer cooperates；The silver staining reagent 2 is mixed by 0.2% silver nitrate and 0.007% benzene sulfonic acid dyeing liquor；
The silver staining reagent 3 is prepared by 2.5% sodium carbonate, 0.1% formaldehyde and 0.002% sodium thiosulfate developer solution；The silver
Transfection reagent 4 forms with 1.0% glacial acetic acid, 5.0% sodium acetate and 10% glycerine terminate liquid.
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| CN201810970225.8A CN109212006A (en) | 2018-08-24 | 2018-08-24 | A kind of unicellular agarose gel electrophoresis kit |
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|---|---|---|---|
| CN201810970225.8A CN109212006A (en) | 2018-08-24 | 2018-08-24 | A kind of unicellular agarose gel electrophoresis kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112223466A (en) * | 2020-10-15 | 2021-01-15 | 东易日盛智能家居科技有限公司 | Method for treating plate without formaldehyde release |
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| CN104120173A (en) * | 2013-04-24 | 2014-10-29 | 温州医学院 | Application of basic fuchsin and derivatives thereof in DNA detection on polyacrylamide gel |
| WO2017091952A1 (en) * | 2015-11-30 | 2017-06-08 | 谢彦晖 | Use of akt2 in diagnosis and treatment of tumor |
| US20170234857A1 (en) * | 2016-02-17 | 2017-08-17 | Androvia Life Sciences LLC | Methods and Test Kits for Determining Male Fertility Status |
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2018
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020198361A1 (en) * | 1997-02-20 | 2002-12-26 | Catherine Rougeot | Therapeutic use of the smr 1 protein and active derivatives thereof |
| CN102586411A (en) * | 2011-01-18 | 2012-07-18 | 温州安得森生物科技有限公司 | Safe and environment-friendly DNA (Deoxyribose Nucleic Acid) silver staining method for polyacrylamide gel |
| CN104120173A (en) * | 2013-04-24 | 2014-10-29 | 温州医学院 | Application of basic fuchsin and derivatives thereof in DNA detection on polyacrylamide gel |
| WO2017091952A1 (en) * | 2015-11-30 | 2017-06-08 | 谢彦晖 | Use of akt2 in diagnosis and treatment of tumor |
| US20170234857A1 (en) * | 2016-02-17 | 2017-08-17 | Androvia Life Sciences LLC | Methods and Test Kits for Determining Male Fertility Status |
Non-Patent Citations (1)
| Title |
|---|
| TREVIGEN: "CometAssay® Silver Kit Reagents for Comet Assay and Staining with Silver Catalog # 4251-050-K", 《HTTPS://TREVIGEN.COM/PRODUCTS-SERVICES/CELL-STRESS-AND-DNA-DAMAGE/DNA-DAMAGE/COMET-ASSAY/CELL-STRESS-AND-DNA-DAMAGE-DNA-DAMAGE-COMET-ASSAY-STANDARD-COMET-ASSAY/SILVER-STAINING-KIT/COMETASSAY-SILVER-KIT/》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112223466A (en) * | 2020-10-15 | 2021-01-15 | 东易日盛智能家居科技有限公司 | Method for treating plate without formaldehyde release |
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