Gwaro reSainzi yeZhizha: Kana Mafungu ekupisa e40°C Akasangana Nekuyedza kweMolecular

Kupisa kwakanyanya kwakaramba kuripo munzvimbo zhinji dzeChina munguva pfupi yapfuura. Musi wa24 Chikunguru, Shandong Provincial Meteorological Observatory yakaburitsa yambiro yekupisa kwakanyanya yeyero, ichifanotaura tembiricha "yakafanana nesauna" ye35-37°C (111-133°F) uye hunyoro hwe80% kwemazuva mana anotevera munzvimbo dziri mukati menyika. Tembiricha munzvimbo dzakadai seTurpan, Xinjiang, dziri kusvika 48°C (111-133°F). Wuhan neXiaogan, Hubei, dziri pasi peyambiro yeorenji, tembiricha dzichipfuura 37°C mune dzimwe nzvimbo. Mukupisa uku kunopisa, nyika iri pasi pemapipettes iri kusangana nekukanganisika kusingawanzoitiki—kugadzikana kwe nucleic acids, kushanda kwema enzymes, uye mamiriro emuviri e reagents zvese zvinokanganiswa chinyararire nekupisa.

Kubviswa kweNucleic acid kwava kumhanya-mhanya nenguva. Kana tembiricha yekunze ikapfuura 40°C, kunyangwe mhepo inotonhodza yakabatidzwa, tembiricha yetafura yekushandisa inowanzo famba pamusoro pe28°C. Panguva ino, RNA samples dzakasiyiwa munzvimbo yakashama dzinoora nekukurumidza kupfuura mumatsutso nemumatsutso. Mukubvisa magineti, mushonga webuffer unozara munzvimbo iyoyo nekuda kwekukurumidza kubhururuka kwe solvent, uye makristaro anowira nyore nyore. Makristaro aya anokonzera kuchinja kukuru mukushanda kwe nucleic acid capture. Kushanduka-shanduka kwe organic solvents kunowedzera panguva imwe chete. Pa30°C, huwandu hwe chloroform volatilization hunowedzera ne40% zvichienzaniswa ne25°C. Munguva yekushanda, zvakakosha kuve nechokwadi chekuti kumhanya kwemhepo mu fume hood kuri 0.5m/s, uye shandisa magirovhosi e nitrile kuchengetedza kushanda kwekudzivirira.

Kuyedza kwePCR kunosangana nekukanganisika kwakanyanya kwetembiricha. Zvinhu zvakaita seTaq enzyme uye reverse transcriptase zvinonzwa zvakanyanya kana tembiricha ikangoerekana yachinja. Kunyungudika pamadziro echubhu mushure mekubviswa mufiriji ye -20°C kunogona kukonzera kurasikirwa kwemabasa eenzyme anopfuura 15% kana ikapinda mu reaction system. Mhinduro dze dNTP dzinogonawo kuratidza kuora kunowanikwa mushure memaminitsi mashanu chete ekushandiswa patembiricha yemukamuri (>30°C). Kushanda kwemidziyo kunokanganiswawo netembiricha yepamusoro. Kana tembiricha yemurabhoritari iri >35°C uye kubviswa kwekupisa kwePCR mudziyo usina kukwana (<50 cm kubva pamadziro), musiyano wetembiricha yemukati unogona kusvika 0.8°C. Kutsauka uku kunogona kukonzera kushanda kwekuwedzera pamucheto weplate ye96-well kudzikira neanopfuura 40%. Mafirita eguruva anofanira kucheneswa nguva dzose (kuunganidzwa kweguruva kunoderedza kushanda kwekupisa ne50%), uye mhepo inotonhorera inofanira kudzivirirwa. Uyezve, paunenge uchiita kuyedza kwePCR husiku hwese, dzivisa kushandisa PCR mudziyo se "makeshift firiji" yekuchengetera sampuro. Kuchengetwa pa4°C kweanopfuura maawa maviri kunogona kukonzera kuumbwa kwemvura mushure mekunge chivharo chinodziya chavharwa, zvichideredza mashandiro emuviri uye zvinogona kukanganisa mamodule esimbi emudziyo.

Kana paine yambiro dzinogara dzichiitika pakupisa kwakanyanya, mamorekuru emurabhoritari anofanirawo kuridza alarm. Masampuli eRNA anokosha anofanira kuchengetwa kumashure kwefiriji ye -80°C, uye mukana wekusvika wakaganhurirwa kune nguva dzekupisa kwakanyanya. Kuvhura gonhi refiriji ye -20°C kanopfuura kashanu pazuva kuchawedzera kushanduka kwekupisa. Midziyo inogadzira kupisa kwakanyanya inoda nzvimbo inosvika 50 cm yekupisa kumativi ese nekumashure. Uyezve, zvinokurudzirwa kugadzirisa nguva yekuedza: 7:00-10:00 AM yekushanda kunonyanya kukanganiswa nekupisa kwakadai sekubviswa kweRNA uye qPCR loading; 1:00-4:00 PM yebasa risiri rekuyedza senge kuongorora data. Iyi nzira inogona kudzivirira zvinobudirira kukwirira kwekupisa kwakanyanya kuti kusapindirane nematanho akakosha.

Kuedza kwemamorekuru panguva yekupisa kunoedza zvese hunyanzvi uye kushivirira. Pasi pezuva rezhizha risingaperi, pamwe inguva yekuisa pipette yako pasi wowedzera bhokisi rechando kune mienzaniso yako kuti mudziyo ubvise kupisa kwakawanda. Kuremekedza uku kushanduka kwekushisa ndiko kwakakosha kwazvo murabhoritari munguva yekupisa kwezhizha—pamusoro pezvose, mukupisa kwe40°C kwezhizha, kunyange mamorekuru anoda "nzvimbo yepasi pevhu" yakachengetedzwa zvakanaka.