Kusamala polyacrylamide gel osakaniza electrophoresis

· polyacrylamidegel osakaniza ayenera ndi acrylamide monoma, polymerization poyambira zakuthupi, chothandizira, ndi ufulu mchere ndi buffer osakaniza pamodzi.

· acrylamidendi BIS (N, N '- methylene double acrylamide) ndi matrix a gel osakaniza.

· ammonium sulfate kuyamba zomatira polymerization ndondomeko. Kupanga guluu kumafuna 10% ammonium sulphate yankho lokonzedwa m'madzi. Zambiri mwazidziwitso zidawonetsa kufunika kogwiritsa ntchito mwachangu. Komabe, yankho la 10% litha kuyikidwa pa 4 ℃ kwa milungu ingapo popanda kutayika kwakukulu kwa ntchito. Pangani mpaka 10 ml ndikutaya pamene guluu likulephera kuphatikiza.

Langizo: Peresenti yaacrylamidepotsata guluu ndi mapuloteni guluu si chimodzimodzi. Ngati mukugwiritsa ntchito premade acrylamide: BIS solution, onetsetsani kuti mwatenga botolo loyenera.

· TEMED (N, N, N ', N'- tetramethyl ethylenediamine) ndi chothandizira, mu botolo la bulauni, loyikidwa mufiriji. Onjezani musanathire guluu.

· Polyacrylamide electrophoresis ntchito galasi electrophoresis ayenera kutsukidwa pamaso ndi pambuyo nthawi iliyonse. Pambuyo pa electrophoresis, sambani ndi burashi yofewa ndi nsalu m'madzi otentha a sopo, muzimutsuka ndi madzi osungunuka ndikuyimirira kuti muume.

· Chinyontho ndi fumbi zitha kuyambitsa ndi polima yopanda kanthu. Pamaso electrophoresis, kuyeretsa galasi mbale ndi galasi zotsukira ndi misozi ndi zofewa burashi. Sambani ndi madzi osungunuka ndikuwumitsa bwino ndi pepala lopukuta. Kutsuka ndi 70% ethanol musanapukute ndi pepala kumathandiza kuyeretsa ndi kufulumizitsa kuyanika. Onjezani zitsanzo za acrylamide motsatizana: BIS, madzi, yankho la buffer, ammonium persulfate, TEMED. Gwirani bwino ndikutsanulira nthawi yomweyo.

Sikuti degass Polyacrylamide pamaso polymerization. (Acrylamide inkaikidwa mu vacuum kuchotsa thovu chifukwa mpweya umalepheretsa polymerization.)

Malangizo a zitsanzo za mfundo zopingasa za glue.

Ikani pepala lakuda m'bokosi lakumunsi, maziko akuda kuti mupange dzenje lachitsanzo kuti muwone bwino.
- thanki yodzaza ndi glue, pamwamba pa colloid.
· ngati m'mphepete muli kuwala, kuyatsa magetsi, lolani kuwala kuwala colloid. Jambulani chitsanzo mu pipette.
· pogwiritsa ntchito chipangizo cha pipetting.
· mu 10-200 mu 1 madzi osuntha malo angagwiritsidwe ntchito pa mfundo zambiri pa chitsanzo. Kwa mabowo ang'onoang'ono (osakwana 10μ1), mutu wautali wa pipette womwe umagwiritsidwa ntchito potsata guluu ndiwosavuta.

· mapaipi kungomizidwa mu chitsanzo, pokoka madzimadzi oyenda pang'onopang'ono. Chitsanzochi chikhoza kuwoneka ngati chomata ndi glycerin, ndipo kupopera kofulumira kumatha kukoka thovu la mpweya mumutu wa pipette.

Zitsanzo pambuyo pokoka mutu wa pipetting, zidzasuntha mutu wamadzimadzi m'mphepete mwa chitoliro kapena kupukuta pepala kumayamwa mutu wamadzi kunja kwa madontho. Samalani kuti musayamwe chitsanzo.

Ikani chitsanzo mu dzenje lachitsanzo

· pipetting chipangizo kusunga kuthamanga pang'ono, kupanga zitsanzo pang'ono kusuntha kusefukira madzi mutu.
· mutu wa chitoliro wolowetsedwa mu chotchinga, chokwera pang'ono kuposa mabowo a malo, sungani kupanikizika kwabwino. Nsonga ya pipette ikhoza kulowetsedwa mu dzenje laling'ono.
· Pang'onopang'ono ndi pang'onopang'ono zitsanzo kunja. Nsonga ya pipette imayikidwa pamwamba pa dzenje lachitsanzo, ndipo chitsanzocho chidzamira mu dzenje. Lolani chitsanzo chimire kuti mudzaze dzenje lachitsanzo m'malo mokankhira mkati.
· dontho lomaliza lachitsanzo lokhala ndi mutu wamadzimadzi, madziwo amasunthira ku mwendo wachiwiri, kukweza pang'onopang'ono kusuntha kwamadzimadzi, kusuntha kuchokera ku buffer.

Kodi vertical glue sampling?

· ofukula zomatira mfundo chitsanzo mabowo anapanga pakati zidutswa ziwiri galasi. Mu guluu woonda kwambiri, mutu wa pipette sungathe ngakhale kuyikidwa pakati pa mbale ziwiri zamagalasi. Penyani glycerin! Ikani mutu wa pipette pa dzenje lachitsanzo ndipo chitsanzocho chidzamira mu dzenje.
· mfundo chitsanzo pamaso, onetsetsani kuika mfundo ofukula wa polypropylene acyl gel osakaniza dzenje ndi nachapidwa woyera. Tsukani acrylamide yopanda polymerized ndi madzi omwe angawoneke pansi pa dzenje lachitsanzo. Madzi amatha kupanga dzenje lachitsanzo kukhala laling'ono kwambiri. Gwiritsani ntchito syringe ya 25ml kapena 50ml ndi singano ya 18-gauge. Thirani mu chotchinga cha electrophoresis ndikutsuka mosamala dzenje lachitsanzo.
· Zingakhale zovuta kuwona dzenje lachitsanzo, koma nthawi yomweyo, zina zonse ndizosavuta. Ngati pali mabowo ochulukirapo, buffer yachitsanzo imatha kuyesedwa ndi bromophenol buluu.