Kariya don polyacrylamide gel electrophoresis

· polyacrylamidegel dole ne ta acrylamide monomer, polymerization fara abu, mai kara kuzari, da hakkin gishiri da kuma cakude buffer tare.

· acrylamideda BIS (N, N '- methylene double acrylamide) shine matrix gel ɗin monomer.

· ammonium persulfate fara m polymerization tsari. Tsarin manne yana buƙatar 10% ammonium persulfate bayani wanda aka shirya cikin ruwa. Yawancin bayanan sun nuna buƙatar amfani mai aiki. Duk da haka, 10% bayani za a iya sanya a 4 ℃ ga dama makonni ba tare da gagarumin asarar aiki. Yi har zuwa ml 10 kuma jefar lokacin da manne ya kasa tarawa.

Tukwici: Kashi naacrylamidea sequencing manne da protein manne ba iri ɗaya ba ne. Idan ana amfani da acrylamide da aka riga aka yi: Maganin BIS, tabbatar da samun kwalbar da ta dace.

TEMED (N, N, N', N'- tetramethyl ethylenediamine) wani abu ne mai kara kuzari, a cikin kwalabe mai launin ruwan kasa, an sanya shi a cikin firiji. Ƙara kafin a zuba manne.

Polyacrylamide electrophoresis da aka yi amfani da gilashin da aka yi amfani da shi a cikin electrophoresis ya kamata a wanke kafin da bayan kowane lokaci. Bayan electrophoresis, wanke tare da goga mai laushi da zane a cikin ruwan zafi mai zafi, kurkura da ruwa mai narkewa kuma tsayawa ya bushe.

· danshi da ƙura na iya haifar da yumbu mai yumbu. Kafin electrophoresis, tsaftace farantin gilashin tare da mai tsabtace gilashi kuma shafa shi da goga mai laushi. A wanke da ruwa mai tsabta kuma a bushe sosai tare da goge takarda. Rinsing tare da 70% ethanol kafin a shafa da takarda yana taimakawa wajen tsaftacewa da saurin bushewa. Ƙara samfurori na acrylamide a jere: BIS, ruwa, maganin buffer, ammonium persulfate, TEMED. Ki girgiza sosai ki zuba nan da nan.

Ba lallai ba ne don cire polyacrylamide kafin polymerization. (Acrylamide ana amfani dashi don sanya shi a cikin injin motsa jiki don cire kumfa saboda oxygen yana hana polymerization.)

Tukwici na manne manne a kwance.

· sanya baƙar takarda a cikin akwatin ƙasa, baƙar fata don yin wani rami don gani a sarari.
· tanki mai cike da manne, kusa da colloid.
· idan gefen yana da haske, kunna fitilun, bari haske ya haskaka colloid. Zana samfurin a cikin pipette.
· amfani da na'urar bututu ta atomatik.
· a cikin 10-200 mu 1 za a iya amfani da wurin motsi na ruwa a yawancin maki akan samfurin. Don ƙananan ramukan samfurin (kasa da 10μ1), dogon pipette shugaban da aka yi amfani da shi don tsara manne ya fi dacewa.

· bututun da aka nutsar da shi a cikin samfurin, yana shakar ruwa mai motsi a hankali. Samfurin na iya zama m tare da glycerin, kuma saurin yin famfo zai iya jawo kumfa na iska a cikin kan pipette.

Samfurori bayan shakar kan pipetting, za su motsa kan ruwa a hankali a gefen bututun ko shafa takarda suna tsotse kan ruwa a waje da ɗigon ruwa. Yi hankali kada ku tsotse samfurin.

Sanya samfurin a cikin ramin samfurin

· Na'urar bututun don kiyaye ɗan matsa lamba, sanya samfuran su ɗan motsa kan ruwa mai kwararowa.
· kan bututun da aka saka a cikin buffer, dan kadan sama da ramukan tabo, yana kula da matsi mai kyau. Ana iya shigar da tip na pipette a cikin ƙaramin rami.
· sannu a hankali samfuran suna fita. An sanya tip ɗin pipette sama da ramin samfurin aya, kuma samfurin zai nutse cikin rami. Bari samfurin ya nutse don cika ramin samfurin maimakon turawa.
Da zarar digo na ƙarshe na samfurin tare da kan ruwa, ruwa zai matsa zuwa ƙafa na biyu, a hankali ya ɗaga ruwan motsi, ya fita daga buffer.

Yadda ake yin samfurin manne a tsaye?

· ramukan samfurin manne a tsaye da aka samu tsakanin gilashin guda biyu. A cikin manne mai bakin ciki sosai, ba za a iya saka kan pipette ba tsakanin faranti biyu na gilashi. Duba glycerin! Sanya shugaban pipette akan ramin samfurin kuma samfurin zai nutse cikin rami.
· batu samfurin kafin, tabbatar da saka a tsaye batu na polypropylene acyl gel samfurin rami ne kurkura da tsabta. A wanke acrylamide maras-polymerized da ruwan da zai iya bayyana a kasan ramin samfurin. Ruwa na iya sa ramin samfurin ya zama ƙarami sosai. Yi amfani da sirinji 25ml ko 50ml da allura mai ma'auni 18. Zuba a cikin buffer electrophoresis kuma a hankali zubar da ramin samfurin.
· yana iya zama da wahala a ga ramin samfurin, amma a daidai wannan, sauran yana da sauƙi. Idan akwai ramuka masu yawa, ana iya gwada buffer samfurin tare da bromophenol blue.