I-PCR FAQ 1. I-false negative, akukho bhanti yokukhulisa ibonakala 2. I-false positive 3. Amabhanti okwandisa angangqalanga avela 4. Imicu yokutsala okanye i-smear strips ivela:
1I-false negative, akukho band yandisiweyo ibonakala Imiba ephambili ye-PCR reaction are
① ukulungiswa kwe-template nucleic acid
② umgangatho wokuqala kunye neenkcukacha
③ umgangatho we-enzyme kunye
④ Iimeko zomjikelezo wePCR.Ukufumana izizathu, uhlalutyo kunye nophando kufuneka kwakhona lwenziwe kula makhonkco angentla.
Isakhelo:
① Ithemplethi iqulethe iiproteni zokungcola
② Ithemplethi iqulethe i-Taq enzyme inhibitors
③ Iiprotheyini ezikwitemplate azityiswa kwaye zisuswe, ngakumbi iihistones kwiichromosomes.
④ Kulahlekile kakhulu ngexesha lokutsalwa kunye nokulungiswa kwetemplate, okanye i-phenol yaphefumlelwa
⑤I-template ye-nucleic acid ayifakwanga ngokupheleleyo.Xa umgangatho we-enzymes kunye ne-primers ulungile, ukuba i-amplification bands ayibonakali, kunokwenzeka ukuba kukho into engalunganga kwinkqubo yokugaya i-specimen okanye inkqubo ye-template nucleic acid extraction.Ngoko ke, isisombululo esisebenzayo nesizinzileyo sokugaya kufuneka silungiswe, kwaye inkqubo kufuneka ilungiswe kwaye akufanele iguqulwe ngokuthanda..Ukungasebenzi kwe-Enzyme: Kuyimfuneko ukutshintsha i-enzyme entsha, okanye usebenzise zombini i-enzymes endala kunye namatsha ngexesha elifanayo ukuhlalutya ukuba izinto ezingalunganga zibangelwa ukulahlekelwa okanye ukungonelanga komsebenzi we-enzyme.Kufuneka kuqatshelwe ukuba ngamanye amaxesha i-enzyme ye-Taq iyalibaleka.I-Primers: umgangatho we-Primer, ukugxininiswa kwe-primer, kunye nokuba ukugxininiswa kwee-primers ezimbini zi-symmetrical zizizathu eziqhelekileyo zokungaphumeleli kwe-PCR okanye i-amplification bands enganelisiyo kunye nokusabalalisa lula.Kukho iingxaki kumgangatho we-primer synthesis kwezinye iibhetshi.Enye yeeprimers ezimbini ine-concentration ephezulu kwaye enye ine-concentration ephantsi, ekhokelela kwi-low-efficiency asymmetric amplification.
Amanyathelo okuchasa ngala:
① Khetha iyunithi elungileyo yokuhlanganisa.
② Ukuxinwa kwee-primers akufanele kujonge kuphela kwixabiso le-OD, kodwa kwakhona ingqalelo kwisisombululo se-primer stock for agarose gel electrophoresis.Kufuneka kubekho i-primer bands, kwaye ukukhanya kwee-primer bands ezimbini kufuneka kufane ngokulinganayo.Ngokomzekelo, enye i-primer inebhendi kwaye enye i-primer ayinayo ibhendi.Kwimicu, i-PCR inokusilela ngeli xesha kwaye kufuneka isonjululwe ngothethwano kunye neyunithi yokudityaniswa kweprimer.Ukuba enye i-primer inokukhanya okuphezulu kwaye enye inokukhanya okuphantsi, linganisa ugxininiso xa uhlambulula iiprimers.
③ Iiprimers kufuneka zigcinwe ekugxininiseni okuphezulu kunye neencinci ezincinci ukukhusela ukukhenkcezwa okuphindaphindiweyo kunye nokunyibilika okanye ukugcinwa kwexesha elide kwisikhenkcisi, oku kunokukhokelela ekudakaleni kunye nokuthotywa kwe-primers.
④Uyilo lwe-primer alunangqiqo, njengokuba ubude be-primer akwanele, i-dimers yenziwe phakathi kwe-primers, njl.Ukuba ugxininiso luphezulu kakhulu, lunokunciphisa ukucaciswa kwe-PCR yokukhulisa.Ukuba i-concentration iphantsi kakhulu, iya kuchaphazela imveliso yokukhulisa i-PCR kwaye ibangele ukuba i-PCR yokukhulisa ingaphumeleli ngaphandle kokukhulisa iibhendi.Utshintsho kwivolumu yokusabela: Ngokuqhelekileyo imiqulu esetyenziselwa ukukhulisa i-PCR yi-20ul, 30ul, kunye ne-50ul.Okanye i-100ul, yiyiphi ivolumu ekufuneka isetyenziswe kwi-PCR yokukhulisa ibekwe ngokweenjongo ezahlukeneyo zophando lwezesayensi kunye novavanyo lweklinikhi.Emva kokwenza umthamo omncinci, onjenge-20ul, kwaye emva koko wenze umthamo omkhulu, kufuneka ulandele imiqathango, ngaphandle koko iya kusilela ngokulula.Izizathu zomzimba: I-Denaturation ibaluleke kakhulu kwi-PCR yokukhulisa.Ukuba ubushushu be-denaturation buphantsi kwaye ixesha le-denaturation lifutshane, izinto ezingalunganga zinokwenzeka kakhulu;ubushushu be-annealing buphantsi kakhulu, obunokubangela ukukhulisa okungangqalanga kunye nokunciphisa ukusebenza kakuhle kokukhulisa.Iqondo lobushushu le-anneal liphezulu kakhulu.Ichaphazela kakhulu ukubotshwa kweeprimers kwiitemplates kwaye inciphisa ukusebenza kakuhle kokukhulisa iPCR.Ngamanye amaxesha kuyimfuneko ukusebenzisa i-thermometer esemgangathweni ukujonga i-denaturation, i-annealing kunye namaqondo okushisa okwandisa kwi-amplifier okanye imbiza e-soluble yamanzi.Esi sesinye sezizathu zokungaphumeleli kwePCR.Umahluko wolandelelwano ekujoliswe kulo: Ukuba ulandelelwano lwethagethi lutshintshwe okanye lucinyiwe, oluchaphazela ukubophelela okuthe ngqo kwe-primer kwi-template, okanye i-primer kunye netemplate ilahlekelwa kulandelelwano oluncedisayo ngenxa yokucinywa kwecandelo elithile lolandelelwano lwethagethi, i-PCR amplification. ayiyi kuphumelela.
2.i-false positive Ibhendi yokukhulisa i-PCR ebonakala ihambelana nebhendi yolandelelwano ekujoliswe kuyo, kwaye ngamanye amaxesha ibhendi inocwangco kwaye iqaqambe ngakumbi.Uyilo olungafanelekanga lwe-primer: Ulandelelwano lokukhulisa olukhethiweyo lune-homology kunye nolandelelwano lwe-amplification engajoliswanga, ngoko ke xa usenza i-PCR amplification, imveliso ye-PCR eyandisiweyo yinkqubo engajoliswanga.Ukuba ulandelelwano ekujoliswe kulo lufutshane kakhulu okanye i-primer imfutshane kakhulu, ii-positives ezingeyonyani zinokuvela ngokulula.Iiprimers kufuneka zenziwe ngokutsha.Ukungcoliswa okunqamlezayo kokulandelelana okujoliswe kuyo okanye iimveliso zokukhulisa: Kukho izizathu ezibini zolu ngcoliseko: Okokuqala, ukungcoliswa kwe-cross-contamination ye-genome yonke okanye amaqhekeza amakhulu, okukhokelela kwiimpawu zobuxoki.Le nto yobuxoki ingasonjululwa ngezi ndlela zilandelayo: Qaphela kwaye ube mnene xa usebenza ukukhusela ulandelelwano olujoliswe kuyo ukuba lufunxe kwisampulu yompu okanye ukutshiza ngaphandle kwetyhubhu ye-centrifuge.Ngaphandle kwee-enzymes kunye nezinto ezingenakukwazi ukumelana nobushushu obuphezulu, zonke ii-reagents okanye izixhobo kufuneka zihlanjululwe ngoxinzelelo oluphezulu.Zonke iityhubhu ze-centrifuge kunye neengcebiso ze-injection pipette yesampuli kufuneka zisetyenziswe kanye.Ukuba kuyimfuneko, iityhubhu zokuphendula kunye ne-reagents zikhanyiswa ngokukhanya kwe-ultraviolet ngaphambi kokuba ungeze umzekelo ukutshabalalisa i-nucleic acids ekhoyo.Okwesibini kukungcoliswa kwamaqhekeza amancinci e-nucleic acids emoyeni.Ezi ziqwenga zincinci zifutshane kunolandelelwano ekujoliswe kulo, kodwa zine-homology ethile.Ziyakwazi ukudityaniswa komnye nomnye, kwaye emva kokuba zincedise kwii-primers, iimveliso ze-PCR zinokunyuswa, okubangela ukuba kubekho iimpawu zobuxoki, ezinokuthi zincitshiswe okanye zipheliswe ngeendlela ze-PCR zendlwane.
3.Iibhendi ze-amplification ezingezizo ngqo zivela Iibhendi ezivela emva kokwandiswa kwe-PCR azihambelani nobungakanani obulindelekileyo, nokuba bukhulu okanye buncinci, okanye zombini iibhendi zokukhulisa ezikhethekileyo kunye neebhendi zokukhulisa ezingazodwa zivela ngaxeshanye.Izizathu zokubonakala kweebhendi ezingezizo zizo: okokuqala, ii-primers azihambelani ngokupheleleyo nolandelelwano olujoliswe kuyo, okanye i-primers aggregate ukwenza i-dimers.Isizathu sesibini kukuba i-ion ye-Mg2 + iphezulu kakhulu, ukushisa kwe-annealing kuphantsi kakhulu, kwaye inani lemijikelezo ye-PCR liphezulu kakhulu.Into yesibini ngumgangatho kunye nobuninzi be-enzyme.Ii-Enzymes ezisuka kweminye imithombo zihlala zithandeka kwiibhendi ezingezizo ezodwa kodwa ii-enzymes ezisuka kweminye imithombo azenzi.Izixa ezigqithisileyo zee-enzyme ngamanye amaxesha zinokukhokelela ekukhuliseni okungangqalanga.Amanyathelo okuthintela abandakanya: ukuyila ngokutsha iiprimers ukuba kuyimfuneko.Nciphisa isixa se-enzyme okanye uyibuyisele omnye umthombo.Ukunciphisa inani lee-primers, ukwandisa inani le template ngokufanelekileyo, kwaye unciphise inani lemijikelezo.Yandisa iqondo lobushushu ngokufanelekileyo okanye usebenzise indlela yobushushu obumbini (i-denaturation kwi-93°C, i-annealing kunye nokwandiswa malunga ne-65°C).
4.I-Flaky drag or smears ibonakala i-PCR yokukhulisa ngamanye amaxesha ibonakala njengeebhanti eziqatyiweyo, iibhendi ezifana neshiti okanye iibhanti ezifana nekhaphethi.Izizathu zihlala zibangelwa yi-enzyme eninzi okanye umgangatho ombi we-enzyme, i-concentration ye-dNTP ephezulu kakhulu, i-Mg2 + i-concentration ephezulu, iqondo lokushisa eliphantsi kakhulu, kunye nemijikelo emininzi kakhulu.Amanyathelo okuthintela abandakanya: ① Nciphisa ubungakanani be-enzayimi, okanye buyisela i-enzyme ngomnye umthombo.②Ukunciphisa ukuxinana kwe-dNTP.Ukunciphisa ngokufanelekileyo i-Mg2 + yoxinaniso.Ukwandisa inani leetemplates kwaye unciphise inani lemijikelezo