· polyacrylamidegel e tlameha ho etsoa ka acrylamide monomer, polymerization ho qala lisebelisoa, catalyst, le tokelo ea letsoai le motsoako oa buffer hammoho.
· acrylamidele BIS (N, N '- methylene double acrylamide) ke mofuta oa monoma oa gel matrix.
· ammonium sulfate qala sekhomaretsi polymerization tshebetso.Ho hlophisoa ha sekhomaretsi ho hloka tharollo ea 10% ea ammonium persulfate e lokiselitsoeng ka metsing.Bongata ba tlhahisoleseling bo bontšitse tlhoko ea tšebeliso e sebetsang.Leha ho le joalo, tharollo ea 10% e ka behoa ho 4 ℃ libeke tse 'maloa ntle le tahlehelo e kholo ea mosebetsi.Etsa ho fihlela ho 10 ml 'me u lahle ha sekhomaretsi se hlōleha ho kopanya.
Keletso: Peresente eaacrylamideka tatellano ea sekhomaretsi le protheine glue ha e tšoane.Haeba u sebelisa premade acrylamide: Tharollo ea BIS, etsa bonnete ba hore u fumana botlolo e nepahetseng.
· TEMED (N, N, N ', N '- tetramethyl ethylenediamine) ke mokelikeli, ka botlolo e sootho, e behiloeng ka sehatsetsing.Eketsa pele o tšela sekhomaretsi.
· Polyacrylamide electrophoresis sebelisoa khalase ka electrophoresis lokela ho hlatsoa pele le ka mor'a nako le nako.Ka mor'a electrophoresis, hlatsoa ka borashe bo bonolo le lesela ka metsi a chesang a sesepa, hlatsoa ka metsi a silafetseng 'me u eme ho omella.
· mongobo le lerōle li ka baka ka polymer e se nang letho.Pele ho electrophoresis, hloekisa poleiti ea khalase ka mochine o hloekisang khalase 'me ue hlakole ka borashe bo bonolo.Hlatsoa ka metsi a hloekisitsoeng 'me u omelle hantle ka ho hlakola pampiri.Ho hlatsoa ka 70% ea ethanol pele u hlakola ka pampiri ho thusa ho hloekisa le ho potlakisa ho omisa.Kenya disampole tsa acrylamide ka tatellano: BIS, metsi, tharollo ea buffer, ammonium persulfate, TEMED.Hlatsoa hantle 'me u tšolle hang-hang.
Ha ho hlokahale ho degass polyacrylamide pele polymerization.(Acrylamide e ne e behoa ka har'a vacuum ho tlosa lipululana hobane oksijene e thibela polymerization.)
Malebela a mohlala a horizontal glue point.
· beha sekhechana sa pampiri e ntšo ka lebokoseng le ka tlase, bokamorao bo botšo ho etsa lesoba la sampole ho bona hantle.
· tanka e tlatsitsoeng ka sekhomaretsi, ka holim'a colloid.
· haeba bohale bo e-na le leseli, khantša mabone, leseli le ke le khanye colloid.Beha mohlala ka pipette.
· ho sebelisa mochine o ikemetseng oa pipetting.
· ho 10-200 mu 1 mokelikeli o tsamaeang ntlha e ka sebelisoa boholo ba lintlha tsa sampole.Bakeng sa masoba a manyane haholo a mohlala (ka tlase ho 10μ1), hlooho e telele ea pipette e sebelisetsoang tatellano ea sekhomaretsi e bonolo haholoanyane.
· ho kenya lipeipi ka ho qoelisoa ka har'a sampuli, ho hema mokelikeli o tsamaeang butle.Mohlala o ka 'na oa bonahala o khomaretse glycerin,' me ho pompa ka potlako ho ka 'na ha hula li-bubble tsa moea hloohong ea pipette.
lisampole ka mor'a ho hema hlooho ea pipetting, e tla tsamaisa hlooho ea mokelikeli butle moeling oa phala kapa pampiri ea ho hlakola e monye hlooho ea mokelikeli ka ntle ho marotholi.Hlokomela hore u se ke ua monya sampole.
Beha mohlala ka mokoting oa mohlala
· pipetting sesebediswa ho boloka khatello e nyenyane, etsa disampole hanyenyane fallela khaphatseha mokelikeli hlooho.
· hlooho ea pipetting e kenngoa ka har'a buffer, e phahame hanyenyane ho feta masoba a matheba, boloka khatello e ntle.Ntlheng ea pipette e ka kenngoa ka sekoting se senyenyane.
· butle-butle le butle-butle lisampole li tsoa.Nōka ea pipette e behoa ka holim'a lesoba la mohlala oa ntlha, 'me sampole e tla teba ka sekoting.Tlohela mohlala ho teba ho tlatsa lesoba la mohlala ho e-na le ho sutumelletsa ka hare.
· hang ha lerotholi la ho qetela la sampole e nang le hlooho ea mokelikeli, mokelikeli o tla ea leotong la bobeli, butle-butle a phahamise mokelikeli, a tlohe ka har'a buffer.
Mokhoa oa ho etsa sampole ea glue e otlolohileng?
· likoti tsa mohlala oa sekhomaretsi tse entsoeng lipakeng tsa likhalase tse peli.Ka sekhomaretsi se tšesaane haholo, hlooho ea pipette e ke ke ea kenngoa pakeng tsa lipoleiti tse peli tsa khalase.Sheba glycerin!Beha hlooho ea pipette holim'a lesoba la mohlala 'me sampole e tla teba ka sekoting.
· lisampole pele, etsa bonnete ba hore u beha ntlha e otlolohileng ea polypropylene acyl gel sample lesoba e hlatsuoa e hloekile.Hlatsoa acrylamide ea unpolymerized le metsi a ka hlahang botlaaseng ba lesoba la sampole.Metsi a ka etsa hore lesoba la sampole le be nyane haholo.Sebelisa serinji ea 25ml kapa 50ml le nale ea 18-gauge.Tšela ka har'a buffer ea electrophoresis 'me u phunye ka hloko lesoba la sampole.
· ho ka ba thata ho bona sekoti sa mohlala, empa ka ho tšoanang, tse ling kaofela li bonolo.Haeba ho na le masoba a feteletseng, buffer ea sampole e ka lekoa ka bromophenol blue.
Nako ea poso: Mar-31-2023