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Njengentsha 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

Ngokushesha1

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).

Isaziso:Ama-primer asanda kuklanywa adinga ukuthuthukisa izimo zokusabela futhi aqinisekise ukunemba, ukunemba kanye nomkhawulo wokuthola wokutholwa ngaphambi kokutholwa kwenani elikhulu.

Isinyathelo sesi-2

Dizayina umthetho olandelwayo wokuhlola

Ngokushesha2

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 ukuze uthole 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.

Isaziso:

Izilawuli ezinhle, ezimbi/ezingenalutho kanye nezilawuli zokulawula zangaphakathi kufanele zisethelwe ukuhlolwa ngakunye ukuze kuhlolwe ukufaneleka kwemiphumela yokuhlolwa.

Ukulungiselela ukuhlolwa

Ngokushesha3

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.

Isaziso:

Khipha imanyuwali futhi uyifunde ngokucophelela, futhi wenze amalungiselelo ngaphambi kokuhlolwa ngokuhambisana ngokuqinile nezidingo zemanuwali.

Isinyathelo sesi-4

Lungiselela isistimu yokusabela ye-PCR

Rapid4

Ngokwephrothokholi yokuhlola, hlanganisa ama-primer, i-H2O, ne-2×PCR mix ngokulinganayo, i-centrifuge futhi usabalalise kushubhu yokusabela ngayinye.

Isaziso:

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

Ngokushesha5

Ngokusebenzisa ubuchwepheshe be-Direct PCR, asikho isidingo senqubo yokuhlanza i-nucleic acid eyisicefe, ithempulethi yesampula ingalungiswa phakathi nemizuzu eyi-10, futhi uhlelo lokusabela lwe-PCR oluhambisanayo lungangezwa.

Isaziso:

Indlela ye-cleavage inomphumela wokutholwa ongcono, futhi umkhiqizo otholiwe ungasetshenziselwa ukusabela kokutholwa okuningi.

Ngokushesha6

5.1: Ukunwetshwa okuqondile kwamaqabunga

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 yokuhlukanisa 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.

Ngokushesha7

Engeza isixazululo se-50ul Buffer P2 neutralization bese uxuba kahle.I-lysate ewumphumela ingasetshenziswa njengesifanekiso futhi yengezwe ohlelweni lokusabela lwe-PCR.

Qaphela: Inani lesifanekiso liphakathi kuka-5-10% wesistimu ye-PCR, futhi akufanele lidlule u-20% (isibonelo, ohlelweni lwe-PCR lwe-20μl, engeza u-1-2μl wesisombululo se-lysis, hhayi ngaphezu kuka-4μl).

Isinyathelo sesi-6

Ukusabela kwe-PCR

Ngokushesha8

Ngemva kokubeka i-centrifuging ishubhu yokusabela ye-PCR, ifakwa ensimbini ye-PCR ukuze ikhulise.

Isaziso:

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

Ngokushesha9

M: 100bp DNA Ladder

1\4: Indlela ye-DNA ehlanzekile

2\5: Indlela ye-PCR eqondile

3\6: Ukulawula okungenalutho

I-QC:

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 wokuhlolwa wofuzo lwangaphandle uthi awunayo, okubonisa ukuthi i-DNA efanelekela ukutholwa kwe-PCR evamile ikhishwa kusampula, futhi kungahlulelwa ukuthi ufuzo lwe-XXX alutholakali kusampula.

C. Umphumela wokuhlolwa wofuzo lwe-endogenous wesampula uthi phozithivu, futhi umphumela wokuhlola wofuzo lwangaphandle uthi phozithivu, okubonisa ukuthi i-DNA efanelekela ukutholwa kwe-PCR evamile ikhishiwe kusampula, futhi isampula ye-DNA iqukethe ufuzo lwe-XXX.Ukuhlolwa kokuqinisekisa kungenziwa ngokuqhubekayo.

Isinyathelo sesi-8

Iziqalo zokutholwa kwedizayini

Ngokushesha10

Ngemuva kokuhlolwa, sebenzisa isisombululo se-sodium hypochlorite esingu-2% kanye nesisombululo se-ethanol esingu-70% ukusula indawo yokuhlola ukuvimbela ukungcoliswa kwemvelo.


Isikhathi sokuthumela: Sep-08-2021