CN109212006A - A kind of unicellular agarose gel electrophoresis kit - Google Patents
A kind of unicellular agarose gel electrophoresis kit Download PDFInfo
- Publication number
- 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
- Authority
- CN
- China
- Prior art keywords
- silver staining
- comet
- staining reagent
- electrophoresis
- unicellular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000246 agarose gel electrophoresis Methods 0.000 title claims abstract description 20
- 208000002109 Argyria Diseases 0.000 claims abstract description 72
- 239000012128 staining reagent Substances 0.000 claims abstract description 67
- 238000001962 electrophoresis Methods 0.000 claims abstract description 38
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 36
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 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 abstract description 21
- 229920001817 Agar Polymers 0.000 claims abstract description 16
- 239000008272 agar Substances 0.000 claims abstract description 16
- 239000006166 lysate Substances 0.000 claims abstract description 16
- 239000011521 glass Substances 0.000 claims abstract description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 229960000583 acetic acid Drugs 0.000 claims description 14
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 claims description 14
- 229940092714 benzenesulfonic acid Drugs 0.000 claims description 14
- 239000000834 fixative Substances 0.000 claims description 14
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 14
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000012362 glacial acetic acid Substances 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 10
- 229920000936 Agarose Polymers 0.000 claims description 9
- 238000004043 dyeing Methods 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 108010039627 Aprotinin Proteins 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 7
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 claims description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 7
- 229960004405 aprotinin Drugs 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 239000004615 ingredient Substances 0.000 claims description 7
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 claims description 7
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 claims description 7
- 108010052968 leupeptin Proteins 0.000 claims description 7
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 7
- 239000011734 sodium Substances 0.000 claims description 7
- 239000001632 sodium acetate Substances 0.000 claims description 7
- 235000017281 sodium acetate Nutrition 0.000 claims description 7
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 6
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 6
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 229920002866 paraformaldehyde Polymers 0.000 claims description 5
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229910052709 silver Inorganic materials 0.000 claims description 4
- 239000004332 silver Substances 0.000 claims description 4
- 238000005336 cracking Methods 0.000 claims description 2
- 239000010977 jade Substances 0.000 claims description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims 1
- 239000012096 transfection reagent Substances 0.000 claims 1
- 230000005778 DNA damage Effects 0.000 abstract description 5
- 231100000277 DNA damage Toxicity 0.000 abstract description 5
- 238000003927 comet assay Methods 0.000 abstract description 4
- 231100000170 comet assay Toxicity 0.000 abstract description 3
- 238000007519 figuring Methods 0.000 abstract description 3
- 239000012634 fragment Substances 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 23
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 23
- 239000003599 detergent Substances 0.000 description 12
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 5
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000012921 fluorescence analysis Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 229940122601 Esterase inhibitor Drugs 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 239000002329 esterase inhibitor Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810970225.8A CN109212006A (en) | 2018-08-24 | 2018-08-24 | A kind of unicellular agarose gel electrophoresis kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810970225.8A CN109212006A (en) | 2018-08-24 | 2018-08-24 | A kind of unicellular agarose gel electrophoresis kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109212006A true CN109212006A (en) | 2019-01-15 |
Family
ID=64989625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810970225.8A Pending CN109212006A (en) | 2018-08-24 | 2018-08-24 | A kind of unicellular agarose gel electrophoresis kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109212006A (en) |
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 |
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 |
-
2018
- 2018-08-24 CN CN201810970225.8A patent/CN109212006A/en active Pending
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/》 * |
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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gedik et al. | Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells | |
| Piette et al. | An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies | |
| Haferkamp et al. | Increased expression of connexin 43 in the overactive neurogenic detrusor | |
| JP2022530140A (en) | Hybridization compositions and methods for preparing and using the compositions. | |
| CN110257483B (en) | Hybridization solution for in situ hybridization, preparation method thereof and detection kit | |
| CN109212006A (en) | A kind of unicellular agarose gel electrophoresis kit | |
| Pilka et al. | Epithelial expression of matrix metalloproteinase‐26 is elevated at mid‐cycle in the human endometrium | |
| US7582435B2 (en) | Messenger RNA profiling: body fluid identification using multiplex reverse transcription-polymerase chain reaction (RT-PCR) | |
| Balasubramanyam et al. | Biomarkers of oxidative stress: methods and measures of oxidative DNA damage (COMET assay) and telomere shortening | |
| JPWO2005035736A1 (en) | Method for removing mucus and cell treatment solution and preservation solution used therefor | |
| EP3559271A1 (en) | Fully automated nucleic acid extraction method for tissue samples | |
| US20230313171A1 (en) | Methods and compositions for removal of rna | |
| RU2472859C1 (en) | Method for formation of dna methylation marker systems | |
| US20070122909A1 (en) | Method of treating cells | |
| CN112575071A (en) | Method for directly performing Sanger sequencing on PCR amplification product without depending on purification | |
| WO2020133590A1 (en) | Method and test kit for quickly preparing monomolecular optical spectrum labeling library | |
| Dubská et al. | Detection of apoptosis in paraffin embedded tissues: the influence of tissue type and fixation | |
| JP6593335B2 (en) | Method for determining differentiation state of stem cell and novel differentiation marker used therefor | |
| TW202507250A (en) | How to use the stain-coated cover slide | |
| EP4019646B1 (en) | Methods and kits for detecting sperm dna fragmentation | |
| Shaler | A multi-enzyme electrophoretic system for the identification of seminal fluid from postmortem specimens | |
| US6231736B1 (en) | Detection of alcaline isophosphatases by electrophoresis | |
| Kempf et al. | Analysis of Cellular EMT States Using Molecular Biology and High Resolution FISH Labeling | |
| Crawford | Development of a Rapid and Efficient Molecular Triage Workflow for Non-Semen Containing Digital Penetration Evidence | |
| Mukherjee et al. | RNA In situ Hybridization |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190115 |
|
| RJ01 | Rejection of invention patent application after publication |