PCR FAQ 1. Tsis muaj qhov tsis zoo, tsis muaj qhov cuam tshuam loj tshwm sim 2. Qhov zoo tsis tseeb 3. Cov kab hluav taws xob tsis tshwj xeeb tshwm sim 4. Flaky drag strips lossis smear strips tshwm:

1False negative, tsis muaj amplified band tshwm Qhov tseem ceeb ntawm cov tshuaj tiv thaiv PCR yog

① kev npaj ntawm template nucleic acid

② primer zoo thiab tshwj xeeb

③ enzyme zoo thiab

④ PCR cov xwm txheej.Txhawm rau nrhiav cov laj thawj, kev tshuaj xyuas thiab kev tshawb fawb yuav tsum tau ua los ntawm cov ntawv txuas saum toj no.

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① Cov qauv muaj cov protein tsis huv

② Cov qauv muaj Taq enzyme inhibitors

③ Cov proteins hauv cov qauv tsis zom thiab tshem tawm, tshwj xeeb tshaj yog cov histones hauv chromosomes

④ Ntau dhau lawm ploj thaum lub sijhawm rho tawm thiab npaj cov qauv, lossis phenol tau nqus

⑤ Cov qauv nucleic acid tsis yog denatured kiag li.Thaum qhov zoo ntawm cov enzymes thiab primers yog qhov zoo, yog tias amplification bands tsis tshwm sim, feem ntau yuav muaj ib yam dab tsi tsis ncaj ncees lawm nrog cov txheej txheem digestion ntawm cov qauv los yog cov qauv nucleic acid extraction txheej txheem.Yog li ntawd, yuav tsum tau npaj cov kev daws teeb meem zoo thiab ruaj khov, thiab cov txheej txheem yuav tsum tau kho thiab yuav tsum tsis txhob hloov pauv ntawm qhov xav tau..Enzyme inactivation: Nws yog ib qho tsim nyog los hloov cov enzymes tshiab, lossis siv ob qho tib si qub thiab tshiab enzymes tib lub sijhawm los tshuaj xyuas seb qhov tsis zoo yog tshwm sim los ntawm kev poob lossis tsis txaus enzymes.Nws yuav tsum raug sau tseg tias qee zaum Taq enzyme tsis nco qab.Primers: Primer zoo, primer concentration, thiab seb qhov concentrations ntawm ob primers yog symmetrical yog vim li cas rau PCR tsis ua hauj lwm los yog tsis txaus siab amplification bands thiab yooj yim diffusion.Muaj teeb meem nrog qhov zoo ntawm primer synthesis nyob rau hauv ib co batch.Ib tug ntawm ob primers muaj ib tug siab concentration thiab lwm yam muaj ib tug tsis tshua muaj concentration, uas ua rau tsis tshua muaj-efficiency asymmetric amplification.

Cov tshuaj tiv thaiv yog:

① Xaiv qhov zoo primer synthesis unit.

② Cov concentration ntawm primers yuav tsum tsis tsuas saib tus nqi OD, tab sis kuj them sai sai rau cov primer Tshuag tov rau agarose gel electrophoresis.Yuav tsum muaj primer bands, thiab brightness ntawm ob primer bands yuav tsum roughly tib yam.Piv txwv li, ib tug primer muaj ib tug band thiab lwm primer tsis muaj band.Rau cov strips, PCR tuaj yeem ua tsis tiav nyob rau lub sijhawm no thiab yuav tsum tau daws los ntawm kev sib tham nrog primer synthesis unit.Yog tias ib qho primer muaj qhov ci ci siab thiab lwm qhov muaj qhov ci qis, sib npaug cov concentrations thaum diluting cov primers.

③ Cov primers yuav tsum tau muab khaws cia rau hauv siab concentration thiab me me kom tsis txhob rov khov thiab thawing los yog cia rau lub sij hawm ntev nyob rau hauv lub tub yees, uas yuav ua rau deterioration thiab degradation ntawm primers.

④ Cov primer tsim tsis tsim nyog, xws li primer ntev tsis txaus, dimers yog tsim ntawm primers, thiab lwm yam.Yog tias qhov concentration siab dhau lawm, nws tuaj yeem txo qhov tshwj xeeb ntawm PCR amplification.Yog tias qhov concentration tsawg dhau, nws yuav cuam tshuam rau PCR amplification tawm los thiab txawm ua rau PCR amplification ua tsis tau yam tsis muaj amplifying bands.Kev hloov pauv hauv cov tshuaj tiv thaiv ntim: Feem ntau cov ntim siv rau PCR amplification yog 20ul, 30ul, thiab 50ul.Los yog 100ul, dab tsi ntim yuav tsum tau siv rau PCR amplification yog teem raws li cov hom phiaj sib txawv ntawm kev tshawb fawb thiab kev soj ntsuam kuaj.Tom qab ua ib lub ntim me me, xws li 20ul, thiab tom qab ntawd ua lub ntim loj, koj yuav tsum ua raws li cov xwm txheej, txwv tsis pub nws yuav ua tsis tau yooj yim.Yog vim li cas lub cev: Denaturation tseem ceeb heev rau PCR amplification.Yog hais tias qhov kub thiab txias denaturation tsawg thiab lub sij hawm denaturation luv luv, qhov tsis zoo tsis zoo yuav tshwm sim;lub annealing kub yog tsawg dhau, uas tuaj yeem ua rau tsis muaj qhov tshwj xeeb amplification thiab txo cov kev ua haujlwm tshwj xeeb.Lub annealing kub yog siab dhau lawm.Muaj feem cuam tshuam rau kev khi ntawm primers rau templates thiab txo PCR amplification efficiency.Qee lub sij hawm nws yog ib qho tsim nyog yuav tsum tau siv tus ntsuas ntsuas kub ntsuas los kuaj xyuas qhov denaturation, annealing thiab extension kub nyob rau hauv lub amplifier los yog dej-soluble lauj kaub.Qhov no kuj yog ib qho laj thawj rau PCR tsis ua haujlwm.Lub hom phiaj sib txawv variation: Yog hais tias lub hom phiaj ua ntu zus los yog tshem tawm, uas cuam tshuam rau qhov tshwj xeeb khi ntawm primer rau lub template, los yog cov primer thiab template poob qhov sib txuas ntxiv vim qhov kev tshem tawm ntawm qee ntu ntawm lub hom phiaj ib ntus, PCR amplification yuav tsis vam meej.

2.false positive Lub PCR amplification band uas zoo nkaus li zoo raws li lub hom phiaj sib lawv liag band, thiab tej zaum lub band yog ntau kom meej meej thiab brighter.Kev tsim qauv tsis tsim nyog: Cov kev xaiv amplification ib ntus muaj homology nrog cov uas tsis yog lub hom phiaj amplification ib ntus, yog li thaum ua PCR amplification, cov khoom lag luam PCR amplified yog cov khoom tsis yog lub hom phiaj.Yog hais tias lub hom phiaj ib ntus luv dhau los yog cov primer luv dhau, qhov tsis zoo yuav tshwm sim tau yooj yim.Primers yuav tsum tau redesigned.Kev sib kis ntawm cov phiaj xwm sib txuas lossis cov khoom lag luam nthuav dav: Muaj ob qho laj thawj rau qhov kev kis kab mob no: Ua ntej, kev sib kis ntawm tag nrho cov genome lossis cov khoom loj, ua rau muaj qhov tsis zoo.Qhov tsis zoo no tuaj yeem daws tau los ntawm cov txheej txheem hauv qab no: Ceev faj thiab maj mam thaum ua haujlwm kom tiv thaiv lub hom phiaj ntawm kev nqus mus rau hauv cov qauv phom los yog txaws tawm ntawm lub raj centrifuge.Tsuas yog cov enzymes thiab cov khoom uas tsis tuaj yeem tiv taus qhov kub thiab txias, tag nrho cov reagents los yog cov khoom siv yuav tsum tau ua kom tsis muaj menyuam los ntawm kev kub siab.Tag nrho cov raj centrifuge thiab cov qauv txhaj tshuaj pipette yuav tsum tau siv ib zaug.Yog tias tsim nyog, cov raj tshuaj tiv thaiv thiab cov tshuaj reagents yog irradiated nrog ultraviolet lub teeb ua ntej ntxiv cov qauv los rhuav tshem cov nucleic acids tam sim no.Qhov thib ob yog kev kis kab mob me me ntawm nucleic acids hauv huab cua.Cov kab me me no luv dua li lub hom phiaj sib lawv liag, tab sis muaj qee yam homology.Lawv tuaj yeem sib txuas rau ib leeg, thiab tom qab ua tiav rau cov primers, cov khoom PCR tuaj yeem nthuav dav, ua rau muaj qhov tsis zoo, uas tuaj yeem txo lossis tshem tawm los ntawm cov txheej txheem PCR nested.

 

3.Non-specific amplification bands tshwm Cov bands uas tshwm sim tom qab PCR amplification yog inconsistent nrog rau qhov yuav tsum tau loj, txawm loj los yog me, los yog ob qho tib si tshwj xeeb amplification bands thiab non-specific amplification bands tshwm sim tib lub sij hawm.Yog vim li cas rau cov tsos ntawm cov tsis tshwj xeeb bands yog: ua ntej, cov primers tsis tag complementary rau lub hom phiaj ib theem zuj zus, los yog cov primers aggregate rau tsim dimers.Qhov thib ob yog vim li cas yog tias Mg2 + ion concentration siab dhau lawm, qhov kub thiab txias annealing tsawg dhau, thiab tus naj npawb ntawm PCR cycles siab dhau lawm.Qhov thib ob yog qhov zoo thiab qhov ntau ntawm cov enzyme.Enzymes los ntawm qee qhov chaw feem ntau ua rau cov kab tsis tshwj xeeb tab sis cov enzymes los ntawm lwm qhov chaw tsis ua.Ntau dhau ntawm cov enzymes qee zaum tuaj yeem ua rau tsis muaj kev nthuav dav.Cov kev tiv thaiv muaj xws li: rov tsim cov primers yog tias tsim nyog.Txo cov enzyme los yog hloov nrog lwm qhov chaw.Txo tus nqi ntawm primers, nce tus nqi ntawm cov qauv tsim nyog, thiab txo cov voj voog.Ua kom qhov kub thiab txias kom tsim nyog lossis siv ob txoj kev ntsuas kub (denaturation ntawm 93 ° C, annealing thiab txuas ntxiv ib ncig ntawm 65 ° C).

 

4.Flaky luag los yog smears tshwm sim PCR amplification qee zaum tshwm li smeared bands, daim ntawv zoo li bands los yog ntaub pua plag zoo li bands.Cov laj thawj feem ntau tshwm sim los ntawm cov enzyme ntau dhau lossis tsis zoo ntawm enzyme, siab dNTP concentration ntau dhau, siab Mg2+ concentration, tsis tshua muaj annealing kub, thiab ntau dhau lawm.Cov kev tiv thaiv muaj xws li: ① Txo cov enzyme, lossis hloov cov enzyme nrog lwm qhov.② Txo cov concentration ntawm dNTP.Tsim nyog txo qhov Mg2 + concentration.Nce tus nqi ntawm cov qauv thiab txo cov voj voog