ISikhokelo seNzululwazi yasehlotyeni: Xa i-40 ° C i-Heat Wave idibana neeMvavanyo zeMolekyuli

Amaqondo obushushu aphezulu athe gqolo kulo lonke elase China mva nje. Ngomhla wama-24 kaJulayi, i-Shandong Provincial Meteorological Observatory yakhupha isilumkiso sobushushu obuphezulu obutyheli, ixela kwangaphambili amaqondo obushushu “afana nesauna” angama-35-37°C (111-133°F) kunye nokufuma okungama-80% kwiintsuku ezine ezilandelayo kwimimandla engaphakathi elizweni. Amaqondo obushushu kwiindawo ezifana neTurpan, eXinjiang, afikelela kuma-48°C (111-133°F). I-Wuhan kunye ne-Xiaogan, e-Hubei, ziphantsi kwesilumkiso esiorenji, kunye namaqondo obushushu angaphezu kwe-37 ° C kwezinye iindawo. Kulo bushushu obutshisayo, ihlabathi le-microscopic elingaphantsi kobuso be-pipettes lifumana ukuphazamiseka okungaqhelekanga-ukuzinza kwe-nucleic acids, umsebenzi we-enzymes, kunye nesimo somzimba we-reagents zonke ziphazamiseka ngokuthula yi-heatwave.

Ukukhutshwa kwe-Nucleic acid kuye kwaba lugqatso oluchasene nexesha. Xa iqondo lobushushu langaphandle lingaphezulu kwama-40°C, kwanange-air conditioner ivuliwe, iqondo lobushushu letafile yokusebenza lidla ngokubhabha ngaphezulu kwe-28°C. Ngeli xesha, iisampulu ze-RNA ezishiywe kwindawo evulekileyo zithomalalisa ngaphezulu kokuphindwe kabini ngokukhawuleza njengasentwasahlobo nasekwindla. Kwi-extraction ye-magnetic bead, isisombululo se-buffer sigcwele ekuhlaleni ngenxa yokuguquka ngokukhawuleza kwe-solvent, kwaye iikristali zihamba ngokulula. Ezi kristale ziya kubangela ukuguquguquka okukhulu ekusebenzeni kokubanjwa kwe-nucleic acid. I-Volatility of organic solvents iyanda ngaxeshanye. Kwi-30°C, isixa sekloroform volatilization inyuka ngama-40% xa kuthelekiswa nama-25°C. Ngethuba lokusebenza, kuyimfuneko ukuqinisekisa ukuba isantya somoya kwi-fume hood yi-0.5m / s, kwaye usebenzise iiglavu ze-nitrile ukugcina ukusebenza kokukhusela.

Imifuniselo ye-PCR ijongene nokuphazamiseka kobushushu obuntsonkothileyo. Iireagents ezifana ne-Taq enzyme kunye ne-reverse transcriptase zinovakalelo olugqithisileyo ekuguquguqukeni kobushushu ngequbuliso. Ukuxinana kwiindonga zetyhubhu emva kokususwa kwi-20°C kwisikhenkcisi kunokubangela ngaphezulu kwe-15% ilahleko yomsebenzi we-enzyme ukuba ingena kwinkqubo yokusabela. Izisombululo ze-dNTP zinokubonisa ukuthotywa okubonakalayo emva kwemizuzu nje emi-5 yokuvezwa kubushushu begumbi (>30°C). Ukusebenza kwesixhobo kwakhona kuphazanyiswa ngamaqondo obushushu aphezulu. Xa ubushushu be-ambient yebhubhoratri i> 35 ° C kunye nokukhutshwa kobushushu besixhobo se-PCR akwanelanga (<50 cm ukusuka eludongeni), umahluko wobushushu wangaphakathi ungafikelela kwi-0.8 ° C. Oku kunxaxha kunokubangela impumelelo yokwandisa kumda we-96-well plate yehle ngaphezulu kwe-40%. Izihluzi zothuli kufuneka zicocwe rhoqo (ukuqokelelwa kothuli kunciphisa ukusebenza kakuhle kokutshabalalisa ubushushu ngama-50%), kwaye umoya othe ngqo kufuneka uphetshwe. Ngaphaya koko, xa usenza imifuniselo ye-PCR ngobusuku, kunqande ukusebenzisa isixhobo se-PCR “njengesikhenkcezisi sokuzenzela” ukugcina iisampulu. Ukugcinwa kwi-4 ° C ngaphezu kweeyure ze-2 kunokubangela ukuba i-condensation yenziwe emva kokuba isivalo esivuthayo sivale, sihlambulule inkqubo yokuphendula kunye nokukwazi ukugqwesa iimodyuli zetsimbi zesixhobo.

Zijongene nezilumkiso eziqhubekayo zobushushu obuphezulu, iilebhu zemolekyuli nazo kufuneka zikhalise isilumkiso. Iisampulu ezixabisekileyo ze-RNA kufuneka zigcinwe ngasemva kwisikhenkcisi esingu -80°C, kwaye ufikelelo luthintelwe kumaxesha obushushu obuphezulu. Ukuvula ucango lwesikhenkcisi-20°C ngaphezu kwamaxesha amahlanu ngosuku kuya kwandisa ukuhla kobushushu. Izixhobo zokuvelisa ukushisa okuphezulu zifuna ubuncinane i-50 cm yendawo yokulahla ubushushu kumacala omabini nangasemva. Ngaphaya koko, kuyacetyiswa ukuba kuhlengahlengiswe ixesha lovavanyo: 7:00-10:00 AM kwimisebenzi ebuthathaka kubushushu obufana nokutsalwa kwe-RNA kunye nokulayishwa kwe-qPCR; I-1: 00-4: 00 PM kumsebenzi ongewona wovavanyo njengohlalutyo lwedatha. Esi sicwangciso sinokuthintela ngokufanelekileyo iincopho eziphezulu zokushisa ekuphazamiseni amanyathelo abalulekileyo.

Uvavanyo lwemolekyuli ngexesha lobushushu luvavanyo lwazo zombini ubuchule kunye nomonde. Ngaphantsi kwelanga elingapheliyo lasehlotyeni, mhlawumbi lixesha lokubeka i-pipette yakho kwaye ungeze ibhokisi eyongezelelweyo yomkhenkce kwiisampuli zakho ukuze uvumele isixhobo ukuba sichithe ubushushu obuninzi. Oku kuhlonipha ukuguquguquka kwamaqondo obushushu ngoyena mgangatho uxabisekileyo welebhu ebudeni beenyanga zasehlotyeni ezigqats’ ubhobhoyi—ngapha koko, kubushushu obungama-40°C ehlotyeni, kwaneemolekyuli zifuna “ummandla wokwenziwa kwencam” ogcinwe ngononophelo.