Uhlolojikelele
Ukuhlonza ngokushesha izitshalo ezishintshashintshayo
Umbhalo/Tong Yucheng
Ukusebenza kokuhlola/u-Han Ying
Umhleli/Wen Youjun
Amagama/1600+
Isikhathi sokufunda esiphakanyisiwe/imizuzu eyi-8-10
Ukuhlonza ngokushesha izitshalo ezishintshashintshayo
Njengomuntu osanda kufika elabhorethri, akuwona umsebenzi omuhle ukuhlola izitshalo ezinhle esixukwini sezitshalo ezinezinga eliphansi lokuguqulwa.Okokuqala, i-DNA kufanele ikhishwe enanini elikhulu lamasampuli ngalinye ngalinye, bese kuthi ufuzo lwangaphandle luzotholwa yi-PCR.Kodwa-ke, imiphumela ivamise ukungabi nalutho namabhendi anezinto ezimbalwa ngezikhathi ezithile, kodwa akunakwenzeka ukunquma ukuthi kukhona yini ukutholwa okugejiwe noma ukutholwa okungamanga..Ingabe akusizi kakhulu ukubhekana nenqubo yokuhlola nemiphumela enjalo?Ungakhathazeki, umfowethu ukufundisa ukuthi ungahlunga kanjani izitshalo ezinhle ezishintshashintshayo kalula nangokunembile.
Isinyathelo 1: Iziqalo zokutholwa kwedizayini
Nquma isakhi sofuzo se-endogenous kanye nofuzo lwangaphandle oluzotholwa ngokuya ngesampula ezohlolwa, bese ukhetha ukulandelana okumele okungu-100-500bp ohlotsheni lomklamo we-primer.Ama-primer amahle angaqinisekisa ukunemba kwemiphumela yokutholwa futhi afinyeze isikhathi sokuthola (bona isithasiselo ukuze uthole iziqalisi zokuthola ezivame ukusetshenziswa).
Qaphela:
Ama-primer asanda kuklanywa adinga ukuthuthukisa izimo zokusabela futhi aqinisekise ukunemba, ukunemba, nomkhawulo wokutholwa wokutholwa ngaphambi kokuthola izinga elikhulu.
Isinyathelo sesi-2:Yakha iphrothokholi yokuhlola
Ukulawula okuhle: Sebenzisa i-DNA ehlanzekile equkethe isiqeshana esiqondiwe njengesifanekiso ukuze unqume ukuthi uhlelo lokusabela kwe-PCR nezimo zijwayelekile yini.
Ukulawula okungalungile/okungenalutho: Sebenzisa isifanekiso se-DNA noma i-ddH2O engaqukethe isiqeshana esiqondiwe njengesifanekiso sokuthola ukuthi ukhona yini umthombo wokungcola ohlelweni lwe-PCR.
Isilawuli sereferensi sangaphakathi: sebenzisa inhlanganisela yokuqala/yokuhlola yofuzo lwesampula oluzohlolwa ukuze uhlole ukuthi isifanekiso singatholwa yi-PCR.
Qaphela:
Izilawuli ezinhle, ezimbi/ezingenalutho kanye nezilawuli zokulawula zangaphakathi kufanele zisethelwe ukuhlolwa ngakunye ukuze kuhlolwe ukufaneleka kwemiphumela yokuhlolwa.
Isinyathelo sesi-3: Ukulungiselela ukuhlolwa
Ngaphambi kokusetshenziswa, bheka ukuthi isixazululo sixubene ngokulinganayo.Uma imvula itholakala, idinga ukuchithwa futhi ixutshwe ngokwemiyalelo ngaphambi kokusetshenziswa.Ingxube ye-2×PCR idinga ukufakwa ngamapayipi futhi ixutshwe ngokuphindaphindiwe ne-micropipette ngaphambi kokusetshenziswa ukuze kugwenywe ukusatshalaliswa kwe-ion okungalingani.
Qaphela:
Khipha imiyalelo futhi uyifunde ngokucophelela, futhi wenze amalungiselelo ngaphambi kokuhlolwa ngokuhambisana ngokuqinile nemiyalelo.
Isinyathelo sesi-4: Lungiselela isistimu yokusabela ye-PCR
Ngokusho kwephrothokholi yokuhlola, hlanganisa ama-primer, u-H2O, 2×PCR mix, centrifuge futhi usabalalise kushubhu yokusabela ngayinye.
Qaphela:
Ekuhlolweni kwesilinganiso esikhulu noma eside, kunconywa ukusebenzisa uhlelo lokusabela lwe-PCR oluqukethe i-UNG enzyme, engagwema ngempumelelo ukungcoliswa kwe-aerosol okubangelwa imikhiqizo ye-PCR.
Isinyathelo sesi-5: Engeza isifanekiso sokusabela
Ukusebenzisa ubuchwepheshe be-Direct PCR, asikho isidingo senqubo yokuhlanza i-nucleic acid eyisicefe.Isifanekiso sesampula singalungiswa phakathi kwemizuzu eyi-10 futhi sengezwe ohlelweni oluhambisanayo lwe-PCR.
Qaphela:
Indlela ye-Lysis inomphumela ongcono wokutholwa, futhi umkhiqizo otholiwe ungasetshenziselwa ukusabela kokutholwa okuningi.
5.1: I-PCR eqondile yamaqabunga
Ngokuhambisana nobukhulu besithombe esikubhukwana, sika izicubu zeqabunga ngobubanzi obungu-2-3mm bese usibeka ohlelweni lokusabela lwe-PCR.
Qaphela: Qinisekisa ukuthi izingcezu zamaqabunga zicwiliswe ngokuphelele kusixazululo sokusabela kwe-PCR, futhi ungangezi izicubu zeqabunga eziningi.
5.2: Indlela yokuhlanza amaqabunga
Sika izicubu zeqabunga ngobubanzi obuyi-5-7mm bese uyibeka kushubhu ye-centrifuge.Uma ukhetha amaqabunga avuthiwe, sicela ugweme ukusebenzisa izicubu zomthambo oyinhloko weqabunga.I-Pipette 50ul Buffer P1 lysate ibe yithubhu ye-centrifuge ukuqinisekisa ukuthi i-lysate ingakwazi ukucwilisa ngokuphelele izicubu zeqabunga, ibeke kumjikelezo oshisayo noma kubhavu wensimbi, futhi i-lyse ku-95 ° C imizuzu engu-5-10.
Engeza isixazululo se-50ul Buffer P2 neutralization bese uxuba kahle.I-lysate ewumphumela ingasetshenziswa njengesifanekiso futhi yengezwe ohlelweni lokusabela lwe-PCR.
Qaphela: Inani lesifanekiso kufanele libe phakathi kuka-5-10% wesistimu ye-PCR, futhi akufanele lidlule u-20% (isibonelo, ohlelweni lwe-PCR engu-20μl, engeza u-1-2μl we-lysis buffer, hhayi ngaphezu kuka-4μl).
Isinyathelo 6:Ukusabela kwe-PCR
Ngemva kokubeka i-centrifuging ishubhu yokusabela ye-PCR, yibeke ensimbini ye-PCR ukuze ikhulise.
Qaphela:
Ukusabela kusebenzisa isifanekiso esingahlanjululwanga sokukhulisa, ngakho-ke inani lemijikelezo yokukhulisa imijikelezo engu-5-10 ngaphezulu kunalapho kusetshenziswa ithempulethi ye-DNA ehlanzekile.
Isinyathelo sesi-7: Ukutholwa kwe-Electrophoresis nokuhlaziywa kwemiphumela
M:100bp DNA Ladder
1\4: Indlela ye-DNA ehlanzekile
2\5: Indlela ye-PCR eqondile
3\6: Ukulawula okungenalutho
Ikhwalithi yokulawula:
Imiphumela yokuhlola yezilawuli ezihlukahlukene ezisethwe kusivivinyo kufanele ihlangabezane nezimo ezilandelayo.Uma kungenjalo, imbangela yenkinga kufanele ihlaziywe, futhi ukuhlolwa kufanele kwenziwe futhi ngemva kokususwa kwenkinga.
Ithebula 1. Imiphumela yokuhlolwa evamile yamaqembu ahlukahlukene okulawula
*Lapho i-plasmid isetshenziswa njengesilawuli esihle, umphumela wokuhlolwa kofuzo ongapheli ungaba unegethivu
Ukwahlulela komphumela:
A. Umphumela wokuhlolwa wofuzo lwe-endogenous lwesampula uthi unegethivu, okubonisa ukuthi i-DNA elungele ukutholwa kwe-PCR evamile ayikwazi ukukhishwa kusampula noma i-DNA ekhishiwe iqukethe ama-PCR reaction inhibitors, futhi i-DNA kufanele ikhishwe futhi.
B. Umphumela wokuhlolwa wofuzo lwe-endogenous wesampula uthi phozithivu, futhi umphumela wokuhlola wofuzo lwangaphandle uthi awunayo, okubonisa ukuthi i-DNA elungele ukutholwa kwe-PCR evamile ikhishwa kusampula, futhi kungahlulelwa ukuthi ufuzo lwe-XXX alutholwa kusampula.
C. Umphumela wokuhlola wofuzo lwe-endogenous wesampula uthi phozithivu, futhi umphumela wokuhlolwa wofuzo lwangaphandle uthi positive, okubonisa ukuthi i-DNA elungele ukutholwa kwe-PCR evamile ikhishiwe kusampula, futhi isampula ye-DNA iqukethe ufuzo lwe-XXX.Ukuhlolwa kokuqinisekisa kungenziwa ngokuqhubekayo.
Isinyathelo sesi-8: Iziqalo zokuthola umklamo
Ngemuva kokuhlolwa, sebenzisa isisombululo se-sodium hypochlorite esingu-2% kanye nesisombululo se-ethanol esingu-70% ukuze usule indawo yokuhlola ukuvimbela ukungcoliswa kwemvelo.
Isithasiselo
Ithebula 2. Iziqalo ezisetshenziswa kakhulu zokuthola i-PCR ejwayelekile yezitshalo ezishintshwe izakhi
Idokhumenti eyisethenjwa:
I-SN/T 1202-2010, Indlela yokutholwa kwe-PCR efanelekile yezithako zezitshalo eziguqulwe ngofuzo ekudleni.
Isimemezelo Somnyango Wezolimo 1485-5-2010, Ukuhlolwa kwezithako zezitshalo ezishintshwe izakhi kanye nemikhiqizo yazo-irayisi i-M12 kanye nokuphuma kwayo.
Isikhathi sokuthumela: Jun-09-2021
