Umhlahlandlela Wesayensi Yasehlobo: Lapho Igagasi Lokushisa Elingu-40°C Lihlangana Nokuhlolwa Kwamamolekyuli

Amazinga okushisa aphezulu aqhubekile kulo lonke elaseShayina muva nje. Ngomhlaka-24 kuJulayi, i-Shandong Provincial Meteorological Observatory ikhiphe isexwayiso sokushisa okuphezulu esiphuzi, sibikezela amazinga okushisa “afana ne-sauna” angu-35-37°C (111-133°F) kanye nomswakama ongu-80% ezinsukwini ezine ezizayo ezindaweni ezingaphakathi nezwe. Amazinga okushisa ezindaweni ezifana neTurpan, eXinjiang, asondela ku-48°C (111-133°F). I-Wuhan ne-Xiaogan, eHubei, zingaphansi kwesexwayiso esiphuzi, kanti amazinga okushisa adlula ku-37°C kwezinye izindawo. Kulokhu kushisa okukhulu, umhlaba omncane ngaphansi kobuso bama-pipettes ubhekene nokuphazamiseka okungavamile—ukuqina kwama-nucleic acid, umsebenzi wama-enzyme, kanye nesimo somzimba sama-reagents konke kuphambukiswa buthule yigagasi lokushisa.

Ukukhishwa kwe-Nucleic acid sekuyinto ephikisana nesikhathi. Lapho izinga lokushisa langaphandle lidlula u-40°C, ngisho noma i-air conditioner ivuliwe, izinga lokushisa letafula lokusebenza livame ukuphakama ngaphezu kuka-28°C. Ngalesi sikhathi, amasampula e-RNA ashiywe evulekile ancipha ngokushesha okuphindwe kabili kunasentwasahlobo nasekwindla. Ekukhishweni kwe-magnetic bead, isixazululo se-buffer sigcwele endaweni ngenxa yokuguquguquka okusheshayo kwe-solvent, futhi amakristalu asheshe akhukhuleke. Lawa makristalu azobangela ukuguquguquka okukhulu ekusebenzeni kahle kokubanjwa kwe-nucleic acid. Ukuguquguquka kwe-organic solvents kuyanda ngasikhathi sinye. Ku-30°C, inani le-chloroform volatilization likhuphuka ngo-40% uma kuqhathaniswa no-25°C. Ngesikhathi sokusebenza, kubalulekile ukuqinisekisa ukuthi isivinini somoya esise-fume hood singu-0.5m/s, futhi usebenzise amagilavu ​​​​e-nitrile ukuze ugcine ukusebenza kahle kokuvikela.

Ukuhlolwa kwe-PCR kubhekene nokuphazamiseka kokushisa okuyinkimbinkimbi nakakhulu. Ama-reagent afana ne-Taq enzyme kanye ne-reverse transcriptase azwela kakhulu ekushintsheni kokushisa okungazelelwe. Ukuminyana ezindongeni zeshubhu ngemva kokususwa efrijini elingu-20°C kungabangela ukulahlekelwa komsebenzi we-enzyme okungaphezu kuka-15% uma kungena ohlelweni lokusabela. Izixazululo ze-dNTP zingabonisa nokuwohloka okubonakalayo ngemva kwemizuzu emi-5 nje yokuchayeka ekushiseni kwegumbi (>30°C). Ukusebenza kwethuluzi nakho kuphazanyiswa amazinga okushisa aphezulu. Lapho izinga lokushisa elise-laboratory lingu->35°C futhi ukusulwa kokushisa kwethuluzi le-PCR kunganele (<50 cm ukusuka odongeni), umehluko wokushisa wangaphakathi ungafinyelela ku-0.8°C. Lokhu kuphambuka kungabangela ukusebenza kahle kokukhulisa emaphethelweni epuleti elingu-96-well ukuthi kwehle ngaphezu kuka-40%. Izihlungi zothuli kufanele zihlanzwe njalo (ukuqongelela uthuli kunciphisa ukusebenza kahle kokushisa ngo-50%), futhi kufanele kugwenywe umoya opholile ngqo. Ngaphezu kwalokho, lapho wenza ukuhlolwa kwe-PCR ubusuku bonke, gwema ukusebenzisa ithuluzi le-PCR “njengesiqandisi sesikhashana” sokugcina amasampula. Ukugcinwa ku-4°C isikhathi esingaphezu kwamahora amabili kungabangela ukwakheka komswakama ngemva kokuvalwa kwesivalo esishisayo, kunciphise uhlelo lokusabela futhi kungonakalisa amamojula ensimbi ensimbi.

Njengoba zibhekene nezixwayiso eziqhubekayo zokushisa okuphezulu, amalabhorethri ama-molecule kufanele futhi ahlabe i-alamu. Amasampula e-RNA ayigugu kufanele agcinwe ngemuva kwefriji engu-80°C, ukufinyelela kukhawulelwe ezikhathini zokushisa okuphezulu. Ukuvula umnyango wefriji engu-20°C izikhathi ezingaphezu kwezihlanu ngosuku kuzokwandisa ukuguquguquka kwezinga lokushisa. Imishini ekhiqiza ukushisa okuphezulu idinga okungenani amasentimitha angu-50 esikhala sokusabalalisa ukushisa kuzo zombili izinhlangothi kanye nezinhlangothi zangemuva. Ngaphezu kwalokho, kunconywa ukuhlela kabusha isikhathi sokuhlola: 7:00-10:00 AM ngemisebenzi ezwela ukushisa njengokukhipha i-RNA kanye nokulayisha kwe-qPCR; 1:00-4:00 PM yomsebenzi ongeyona eyokuhlola njengokuhlaziywa kwedatha. Leli cebo lingavimbela ngempumelelo ukuphakama kwezinga lokushisa eliphezulu ukuthi kungaphazamisi izinyathelo ezibalulekile.

Ukuhlolwa kwama-molecule ngesikhathi sokushisa kuyisilingo sobuchwepheshe kanye nokubekezela. Ngaphansi kwelanga lasehlobo elingapheli, mhlawumbe sekuyisikhathi sokubeka phansi i-pipette yakho bese ufaka ibhokisi elengeziwe leqhwa kumasampula akho ukuze ithuluzi likhiphe ukushisa okwengeziwe. Lokhu kuhlonipha ukuguquguquka kwezinga lokushisa kuyikhwalithi yelabhoratri eyigugu kakhulu phakathi nezinyanga zasehlobo ezishisayo—phela, ekushiseni kwehlobo okungu-40°C, ngisho nama-molecule adinga “indawo ye-polar yokwenziwa” eqashwe ngokucophelela.