Primer tsim hauv paus (99% cov teeb meem tuaj yeem daws tau)
1. Primer ntev: Phau ntawv yuav tsum muaj 15-30bp, feem ntau yog li 20bp.Qhov tseeb yog qhov zoo dua yog 18-24bp los xyuas kom meej qhov tshwj xeeb, tab sis ntev dua qhov zoo dua, ntev primer kuj yuav txo tau qhov tshwj xeeb, thiab txo qhov tawm los.
2. Primer amplification span: 200-500bp yog qhov tsim nyog, thiab cov tawg tuaj yeem nthuav dav mus rau 10kb nyob rau hauv cov xwm txheej tshwj xeeb.
3. Primer puag: Cov ntsiab lus ntawm G + C yuav tsum yog 40-60%, G + C amplification nyhuv me ntsis tsis zoo, ntau dhau G + C yog ib qho yooj yim los tshwm tsis tshwj xeeb bands.ATGC yog qhov zoo tshaj plaws faib random, zam cov pawg ntau dua 5 purine lossis pyrimidine nucleotides.Multi-gc rau 5′ kawg thiab nruab nrab ib ntus kom muaj kev ruaj ntseg, zam kev nplua nuj GC ntawm 3′ kawg, tsis muaj GC rau 3 lub hauv paus kawg, lossis tsis muaj GC rau 3 ntawm 5 lub hauv paus kawg.
4. Tsis txhob siv cov txheej txheem thib ob hauv cov primers, thiab tsis txhob sib ntxiv ntawm ob lub primers, tshwj xeeb tshaj yog kev sib txuas ntawm 3 'kawg, txwv tsis pub primer dimer yuav raug tsim thiab tsis yog ib qho tshwj xeeb amplified bands yuav raug tsim.
5. Lub hauv paus ntawm 3 'qhov kawg ntawm primers, tshwj xeeb tshaj yog lub hauv paus kawg thiab penultimate, yuav tsum tau ua ke nruj me ntsis kom tsis txhob muaj PCR tsis ua hauj lwm vim qhov tsis sib xws ntawm lub davhlau ya nyob twg hauv paus.
6. Primers muaj lossis tuaj yeem muab ntxiv nrog qhov chaw cleavage tsim nyog, thiab cov phiaj xwm nthuav dav yuav tsum zoo dua muaj qhov chaw cleavage tsim nyog, uas yog qhov zoo heev rau kev soj ntsuam cleavage lossis molecular cloning.
7. Qhov tshwj xeeb ntawm primers: primers yuav tsum tsis muaj qhov pom tseeb homology nrog rau lwm cov sequences nyob rau hauv lub nucleic acid sequence database.
8. Kawm siv software: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Qhov kev tsim online no ua haujlwm zoo tshaj).
Cov ntsiab lus saum toj no tuaj yeem daws yam tsawg kawg 99% ntawm cov teeb meem tsim primer.
Tswj cov ntsiab lus ntawm primer tsim
1. Primer ntev
General primer ntev yog 18 ~ 30 puag.Feem ntau, qhov tseem ceeb tshaj plaws uas txiav txim siab qhov kub thiab txias ntawm cov primer yog qhov ntev ntawm cov primer.Lub annealing kub ntawm primer feem ntau xaiv (Tm tus nqi -5 ℃), thiab qee qhov ncaj qha siv Tm tus nqi.Cov qauv hauv qab no tuaj yeem siv los ntsuas qhov ntsuas kub ntawm cov primers.
Thaum qhov ntev ntawm primer tsawg dua 20bp: [4 (G + C) + 2 (A + T)] -5 ℃
Thaum qhov ntev ntawm primer ntau dua 20bp: 62.3 ℃ + 0.41 ℃ (% GC) -500 / ntev - 5 ℃
Tsis tas li ntawd, ntau lub software kuj tuaj yeem siv los ntsuas qhov kub thiab txias, lub hauv paus ntsiab lus ntawm kev suav yuav txawv, yog li qee zaum tus nqi suav yuav muaj qhov sib txawv me me.Txhawm rau ua kom zoo rau PCR cov tshuaj tiv thaiv, cov primers luv tshaj plaws uas ua kom qhov kub thiab txias tsis tsawg dua 54 ℃ yog siv rau qhov ua tau zoo thiab qhov tshwj xeeb.
Zuag qhia tag nrho, qhov tshwj xeeb primer nce los ntawm qhov tseem ceeb ntawm plaub rau txhua qhov ntxiv nucleotide, yog li ntawd qhov tsawg kawg nkaus primer ntev rau feem ntau yog 18 nucleotides.Lub sab sauv txwv ntawm primer ntev tsis tseem ceeb heev, feem ntau muaj feem xyuam rau cov tshuaj tiv thaiv efficiency.Vim hais tias ntawm entropy, ntev lub primer, qhov qis dua tus nqi uas nws anneals los khi rau lub hom phiaj DNA los tsim ib tug ruaj khov ob-stranded template rau DNA polymerase los khi.
Thaum siv software los tsim primers, qhov ntev ntawm primers tuaj yeem txiav txim siab los ntawm TM tus nqi nyob rau hauv lem, tshwj xeeb tshaj yog rau primers ntawm fluorescence quantitative PCR, TM = 60 ℃ los yog li ntawd yuav tsum tau tswj.
2.GC cov ntsiab lus
Feem ntau, cov ntsiab lus ntawm G + C hauv cov txheej txheem primer yog 40% ~ 60%, thiab GC cov ntsiab lus thiab Tm tus nqi ntawm ib khub ntawm primers yuav tsum tau sib koom ua ke.Yog tias tus primer muaj qhov hnyav GC lossis AT nyiam, qhov tsim nyog ntawm A, T lossis G thiab C tus Tsov tus tw tuaj yeem ntxiv rau 5 'kawg ntawm primer.
3. Annealing kub
Lub annealing kub yuav tsum yog 5 ℃ qis dua qhov kub tsis tau.Yog hais tias tus naj npawb ntawm primer bases me me, qhov kub thiab txias tuaj yeem nce qhov tsim nyog, uas tuaj yeem nce qhov tshwj xeeb ntawm PCR.Yog tias tus naj npawb ntawm cov hauv paus loj, qhov kub ntawm qhov annealing tuaj yeem raug txo kom tsim nyog.Qhov sib txawv ntawm qhov sib txawv ntawm qhov sib txawv ntawm ib khub ntawm primers ntawm 4 ℃ ~ 6 ℃ yuav tsis cuam tshuam rau PCR tawm los, tab sis qhov zoo tshaj plaws qhov kub ntawm ib khub ntawm primers yog tib yam, uas tuaj yeem sib txawv ntawm 55 ℃ ~ 75 ℃.
4. Zam qhov chaw nruab nrab ntawm cov qauv amplification
Nws yog qhov zoo tshaj plaws kom tsis txhob muaj cov qauv theem nrab ntawm lub template thaum xaiv cov khoom tawg yooj yim.Cov qauv nruab nrab ruaj khov ntawm lub hom phiaj fragment tuaj yeem kwv yees thiab kwv yees los ntawm cov khoos phis tawj cuam tshuam, uas yuav pab tau rau kev xaiv template.Cov txiaj ntsig kev sim qhia tau hais tias qhov kev nthuav dav feem ntau ua tsis tiav thaum lub zog dawb (△G) ntawm thaj av yuav nthuav dav tsawg dua 58.6lkJ / mol.
5. Tsis sib haum nrog lub hom phiaj DNA
Thaum lub hom phiaj DNA loj zuj zus tuaj, cov primer tuaj yeem khi rau ntau qhov chaw ntawm lub hom phiaj DNA, uas ua rau muaj ntau pawg tshwm sim hauv qhov tshwm sim.Lub sijhawm no nws yog qhov yuav tsum tau siv BLAST software kuaj, lub vev xaib:http://www.ncbi.nlm.nih.gov/BLAST/.Xaiv Align ob ntu (bl2seq).
Pasting primer sequences mus rau thaj tsam 1 thiab lub hom phiaj DNA sib lawv liag rau cheeb tsam 2 yog sib hloov, thiab BLAST xam complementary, antisense, thiab lwm yam muaj peev xwm, yog li ntawd cov neeg siv tsis tas yuav tsum tau ceeb toom seb ob chains yog kev nkag siab chains.Koj tuaj yeem nkag mus rau tus lej GI yog tias koj paub tus lej GI ntawm qhov sib lawv liag hauv cov ntaub ntawv, yog li koj tsis tas yuav muab tshuaj txhuam ntau ntawm cov kab ke.Thaum kawg, nyem Align ntawm 3 kom pom tias cov primer muaj ntau qhov chaw homologous hauv lub hom phiaj DNA.
6. Primer davhlau ya nyob twg
Lub 3 'kawg ntawm cov primer yog qhov chaw txuas ntxiv pib, yog li nws yog ib qho tseem ceeb kom tsis txhob muaj qhov tsis sib xws los ntawm qhov pib.Qhov 3 'kawg yuav tsum tsis txhob ntau tshaj 3 sib law liag G lossis C, vim qhov no yuav ua rau cov primer ua yuam kev hauv thaj tsam G + C enrichment sequence.Lub 3 'kawg tsis tuaj yeem tsim cov qauv thib ob, tshwj tsis yog hauv PCR tshwj xeeb (AS-PCR) cov tshuaj tiv thaiv, 3' kawg ntawm cov primer tsis tuaj yeem ua tsis zoo.Piv txwv li, yog hais tias lub encoding cheeb tsam yog amplified, lub 3 'kawg ntawm primer yuav tsum tsis txhob raug txiav nyob rau hauv lub thib peb txoj hauj lwm ntawm codon, vim hais tias qhov thib peb txoj hauj lwm ntawm codon yog nquag degenerate, uas yuav cuam tshuam rau qhov tshwj xeeb thiab efficiency ntawm amplification.Thaum siv annexation primers, xa mus rau lub rooj siv codon, xyuam xim rau kev nyiam lom neeg, tsis txhob siv annexation primers ntawm 3′ kawg, thiab siv ntau dua concentration ntawm primers (1uM-3uM).
7. Secondary qauv ntawm primers
Cov primers lawv tus kheej yuav tsum tsis txhob muaj kev sib txuas ua ke, txwv tsis pub cov primers lawv tus kheej yuav quav rau hauv cov qauv plaub hau, thiab cov qauv thib ob no yuav cuam tshuam rau kev khi ntawm primers thiab templates vim muaj kev cuam tshuam tsis zoo.Yog tias kev txiav txim siab siv dag zog, qhov txuas ntxiv txuas ntxiv ntawm cov primers lawv tus kheej yuav tsum tsis txhob ntau dua 3bp.Yuav tsum tsis muaj qhov sib ntxiv ntawm ob lub primers, tshwj xeeb tshaj yog qhov sib tshooj ntawm 3 'kawg yuav tsum tau zam kom tsis txhob muaj qhov tsim ntawm primer dimers.Feem ntau, yuav tsum tsis pub ntau tshaj 4 lub hauv paus sib law liag los yog kev sib txuam ntawm ib khub ntawm primers.
8. Ntxiv cov cim lossis loci
Lub 5 'kawg muaj kev cuam tshuam me ntsis ntawm kev nthuav dav tshwj xeeb thiab tuaj yeem hloov kho yam tsis muaj kev cuam tshuam rau amplification tshwj xeeb.Kev hloov kho ntawm primer 5 'kawg suav nrog: ntxiv enzyme txwv qhov chaw;Labeled biotin, fluorescence, digoxin, Eu3+, thiab lwm yam. Qhia cov protein khi DNA ua ntu zus;Qhia qhov chaw hloov pauv, ntxig thiab ploj qhov kev hloov pauv tsis tu ncua thiab nthuav qhia cov txheej txheem txhawb nqa, thiab lwm yam. Cov hauv paus ntxiv yuav cuam tshuam ntau dua los yog tsawg dua qhov ua tau zoo ntawm kev nthuav dav thiab ua rau kom muaj peev xwm ntawm primer dimer tsim, tab sis qee qhov kev pom zoo yuav tsum tau ua rau cov kauj ruam tom ntej.Ntxiv cov sequences uas tsis muaj nyob rau ntawm lub hom phiaj ib ntus, xws li kev txwv qhov chaw thiab kev txhawb nqa ib ntus, tuaj yeem ntxiv rau 5' kawg ntawm cov primer yam tsis muaj kev cuam tshuam tshwj xeeb.Cov kab ke no tsis suav nrog hauv kev suav ntawm primer Tm qhov tseem ceeb, tab sis yuav tsum tau sim rau kev sib xyaw ua ke thiab cov qauv hauv nruab nrab.
9. Subclones
Feem ntau, PCR tsuas yog ua ntej cloning, thiab tom qab ntawd peb yuav tsum tau subclone lub hom phiaj tawg mus rau ntau yam vectors, yog li peb yuav tsum tsim cov hauv paus ntxiv rau kev ua haujlwm tom ntej hauv PCR kauj ruam.
Qee cov kab ke tsim los rau subcloning tau sau tseg hauv qab no.
Kev txwv endonuclease txwv qhov chaw tau ntxiv
Ntxiv qhov chaw txwv enzyme yog txoj kev siv ntau tshaj plaws rau subcloning PCR cov khoom.Feem ntau, qhov chaw cleavage yog rau lub hauv paus, ntxiv rau 5 'qhov kawg ntawm qhov chaw cleavage yuav tsum tau ntxiv 2 ~ 3 lub hauv paus tiv thaiv.Txawm li cas los xij, tus naj npawb ntawm cov hauv paus tiv thaiv xav tau los ntawm cov enzymes sib txawv.Piv txwv li, SalⅠ yuav tsum tsis muaj kev tiv thaiv puag, EcoRⅤ xav tau 1 lub hauv paus tiv thaiv, Tsis yog yuav tsum muaj 2 lub hauv paus tiv thaiv, thiab Hind Ⅲ yuav tsum muaj 3 lub hauv paus tiv thaiv.
LIC ntxiv tus Tsov tus tw
Lub npe tag nrho ntawm LIC yog Ligation-Independent cloning, ib txoj kev cloning tsim los ntawm Navogen tshwj xeeb rau nws ib feem ntawm pET vector.PET cov neeg nqa khoom npaj los ntawm LIC txoj kev tsis muaj qhov sib ntxiv ntawm 12-15 lub hauv paus ib qho strand nplaum kawg, uas ntxiv rau qhov sib txuas nplaum kawg ntawm lub hom phiaj ntxig ntu.Rau lub hom phiaj amplification, lub primer 5' ib ntus ntawm cov khoom seem yuav tsum ntxiv rau LIC vector.Lub 3′ → 5′ extranect kev ua ntawm T4 DNA polymerase tuaj yeem tsim ib qho strand nplaum kawg ntawm cov khoom seem tom qab lub sijhawm luv luv.Vim hais tias cov khoom tsuas yog tsim los ntawm kev sib nrig sib annealing ntawm cov npaj cov khoom seem thiab cov vector, txoj kev no yog ceev heev thiab npaum, thiab nws yog qhia cloning.
Qhia TA clone ntxiv tus Tsov tus tw
TA cloning tsis tuaj yeem tsom qhov tawg mus rau hauv vector, yog li tom qab Invitrogen tau qhia txog vector uas tuaj yeem tsom cloning, uas muaj plaub lub hauv paus tseem ceeb GTGGS ntawm ib kawg.Yog li ntawd, nyob rau hauv kev tsim ntawm PCR primers, complementary sequences yuav tsum tau ntxiv raws li, yog li ntawd fragments yuav "taw qhia".
Yog tias koj luv luv rau lub sijhawm, koj tuaj yeem sim ncaj qha synthesis, sib txuas cov noob nrog cov vector, uas yog qhov peb hu ua ET gene synthesis hauv musecularists.
D. In-Fusion cloning txoj kev
Tsis muaj ligase yuav tsum tau, tsis muaj cov tshuaj tiv thaiv ntev.Ntev npaum li qhov ua tiav ntawm ob qho kawg ntawm cov kab hluav taws xob kab hluav taws xob tau qhia Hauv kev tsim cov primers, tom qab ntawd cov khoom PCR thiab linearized vector ntxiv rau hauv in-fusion enzyme tov uas muaj BSA thiab muab tso rau hauv chav tsev kub rau ib nrab ib teev, qhov kev hloov pauv tuaj yeem ua tau.Txoj kev no yog tshwj xeeb tshaj yog haum rau loj ntim hloov dua siab tshiab.
10. Ua ke primer
Qee lub sij hawm, tsuas yog cov ntaub ntawv tsuas yog ib ntus paub txog kev tsim primer.Piv txwv li, yog tias tsuas yog cov amino acid sib lawv liag paub, lub merging primer tuaj yeem tsim.Ib qho kev sib koom ua ke primer yog qhov sib xyaw ntawm cov kab sib txawv uas sawv cev rau txhua qhov sib txawv hauv paus muaj peev xwm uas encode ib qho amino acid.Txhawm rau kom muaj qhov tshwj xeeb, koj tuaj yeem xa mus rau lub rooj siv codon kom txo cov kev sib txuas ua ke raws li kev siv lub hauv paus nyiam ntawm cov kab mob sib txawv.Hypoxanthine tuaj yeem ua ke nrog txhua lub hauv paus kom txo tau qhov kub thiab txias ntawm primer.Tsis txhob siv lub hauv paus txuas ntxiv ntawm 3′ kawg ntawm cov primer vim hais tias qhov annealing ntawm 3 lub hauv paus kawg ntawm 3′ kawg yog txaus los pib PCR ntawm qhov chaw tsis ncaj ncees lawm.Higher primer concentrations (1μM rau 3μM) yog siv vim primers nyob rau hauv ntau annexation mixs tsis yog tshwj xeeb rau lub hom phiaj template.
PCR raw khoomtswj
1. Primer kom muaj nuj nqis
Qhov concentration ntawm txhua tus primer yog 0.1 ~ 1umol lossis 10 ~ 100pmol.Nws yog qhov zoo dua los tsim cov txiaj ntsig xav tau nrog tus nqi qis tshaj ntawm primer.High concentration ntawm primer yuav ua rau mismatch thiab non-specific amplification, thiab ua rau kom lub sij hawm ntawm tsim dimers ntawm primers.
2. Primer concentration
Qhov concentration ntawm primers cuam tshuam qhov tshwj xeeb.Qhov zoo tshaj plaws primer concentration yog feem ntau ntawm 0.1 thiab 0.5μM.Higher primer concentrations ua rau amplification ntawm nonspecific khoom.
3. Annealing kub ntawm primer
Lwm qhov tseem ceeb parameter rau primers yog melting kub (Tm).Qhov no yog qhov kub thiab txias thaum 50% ntawm primers thiab complementary sequences sawv cev raws li ob-stranded DNA molecules.Tm yog yuav tsum tau teem PCR annealing kub.Qhov zoo tshaj plaws, qhov kub ntawm qhov annealing yog tsawg txaus los xyuas kom meej annealing zoo ntawm cov primers nrog lub hom phiaj sib lawv liag, tab sis siab txaus los txo cov nonspecific binding.Tsim nyog annealing kub ntawm 55 ℃ to 70 ℃.Annealing kub yog feem ntau teem 5 ℃ qis dua Tm ntawm primer.
Muaj ntau ntau cov qauv rau kev teeb tsa Tm, uas sib txawv heev nyob ntawm cov qauv siv thiab qhov sib lawv liag ntawm primers.Vim tias feem ntau cov qauv muab qhov kwv yees Tm tus nqi, tag nrho cov qhov kub thiab txias tsuas yog qhov pib.Qhov tshwj xeeb tuaj yeem txhim kho los ntawm kev txheeb xyuas ntau qhov kev cuam tshuam uas nce qhov kub ntawm qhov annealing.Pib hauv qab qhov kwv yees Tm-5 ℃, thiab maj mam nce qhov kub thiab txias hauv qhov nce ntawm 2 ℃.Kev kub siab annealing yuav txo qhov tsim ntawm primer dimers thiab cov khoom tsis yog tshwj xeeb.Rau cov txiaj ntsig zoo tshaj plaws, ob lub primers yuav tsum muaj kwv yees li Tm qhov tseem ceeb.Yog tias qhov sib txawv ntawm Tm ntawm primer khub yog ntau tshaj 5 ℃, cov primers yuav qhia qhov tsis tseeb tseem ceeb pib los ntawm kev siv qhov kub thiab txias hauv lub voj voog.Yog hais tias ob lub primers Tm txawv, teem lub annealing kub rau 5 ℃ qis tshaj qhov qis tshaj Tm.Xwb, txhawm rau nce qhov tshwj xeeb, tsib lub voj voog tuaj yeem ua tau ua ntej ntawm qhov kub thiab txias tsim kom siab dua Tm, ua raws li cov voj voog seem ntawm qhov ntsuas kub uas tsim los rau qis Tm.Qhov no tso cai rau ib feem ntawm daim ntawv lo lus uas peb tau txais nyob rau hauv cov xwm txheej nruj.
4. Primer purity thiab stability
Tus qauv purity ntawm kev cai primers yog txaus rau feem ntau PCR daim ntaub ntawv.Kev tshem tawm ntawm benzoyl thiab isobutylyl pawg los ntawm desalting yog tsawg thiab yog li tsis cuam tshuam nrog PCR.Qee daim ntawv thov yuav tsum tau ua kom huv huv kom tshem tawm ib qho uas tsis yog tag nrho cov kab ke hauv cov txheej txheem synthesis.Cov ntu ntu ntu no tshwm sim vim qhov ua tau zoo ntawm DNA synthesis chemistry tsis yog 100%.Qhov no yog cov txheej txheem ncig uas siv cov tshuaj tiv thaiv rov qab vim txhua lub hauv paus tau ntxiv los ua DNA ntawm 3′ txog 5′.Koj tuaj yeem ua tsis tau hauv ob lub voj voog.Cov primers ntev dua, tshwj xeeb tshaj yog cov ntau dua 50 lub hauv paus, muaj qhov sib faib loj ntawm cov ntu ntu thiab yuav tsum tau ua kom huv.
Cov txiaj ntsig ntawm primers yog cuam tshuam los ntawm kev ua haujlwm ntawm hluavtaws chemistry thiab purification txoj kev.Cov tuam txhab biopharmaceutical, xws li Cytology thiab Shengong, txhua tus siv OD chav tsev yam tsawg kawg nkaus kom ntseeg tau tias tag nrho cov zis ntawm oligonucleoside.Kev cai primers raug xa mus rau hauv cov hmoov qhuav qhuav.Nws yog qhov zoo tshaj plaws los daws cov primers hauv TE kom qhov kawg concentration yog 100μM.TE zoo dua li dej deionized vim tias pH ntawm cov dej feem ntau yog acidic thiab yuav ua rau hydrolysis ntawm oligonucleosides.
Lub stability ntawm primers nyob ntawm qhov chaw cia.Cov hmoov qhuav thiab yaj primers yuav tsum khaws cia ntawm -20 ℃.Primers yaj hauv TE ntawm qhov siab tshaj 10μM tuaj yeem ruaj khov ntawm -20 ℃ rau 6 lub hlis, tab sis tsuas yog khaws cia hauv chav sov (15 ℃ txog 30 ℃) tsawg dua 1 lub lis piam.Cov hmoov qhuav qhuav tuaj yeem khaws cia ntawm -20 C rau tsawg kawg 1 xyoos thiab hauv chav sov (15 C txog 30 C) txog li 2 lub hlis.
5. Enzymes thiab lawv cov concentrations
Tam sim no, Taq DNA polymerase siv yog lub hauv paus gene engineering enzyme synthesized los ntawm cov kab mob coliform.Tus nqi ntawm enzyme xav tau los ua kom cov tshuaj tiv thaiv PCR raug kwv yees li 2.5U (xws li tag nrho cov tshuaj tiv thaiv ntim ntawm 100ul).Yog tias qhov concentration siab dhau lawm, nws tuaj yeem ua rau tsis muaj qhov tshwj xeeb amplification;yog tias qhov concentration tsawg dhau, tus nqi ntawm cov khoom hluavtaws yuav raug txo.
6. Zoo thiab concentration ntawm dNTP
Qhov zoo ntawm dNTP yog ze ze rau qhov concentration thiab kev ua haujlwm ntawm PCR amplification.dNTP hmoov yog granular, thiab nws qhov kev hloov pauv poob nws txoj haujlwm lom neeg yog tias nws khaws cia tsis raug.dNTP cov tshuaj yog acidic, thiab yuav tsum tau siv nyob rau hauv siab concentration, nrog 1M NaOH los yog 1M Tris.HCL buffer tov los kho nws PH rau 7.0 ~ 7.5, me me ntawm sub-packaging, khov cia ntawm -20 ℃.Ntau khov-thawing yuav degrade dNTP.Hauv PCR cov tshuaj tiv thaiv, dNTP yuav tsum yog 50 ~ 200umol / L.Tshwj xeeb, mloog yuav tsum tau them rau qhov concentration ntawm plaub DNTPS yuav tsum sib npaug (sib npaug mole npaj).Yog tias qhov concentration ntawm ib qho ntawm lawv txawv ntawm lwm tus (siab dua lossis qis dua), kev tsis sib haum yuav tshwm sim.Cov concentration tsawg dhau yuav txo cov txiaj ntsig ntawm cov khoom PCR.dNTP tuaj yeem ua ke nrog Mg2+ thiab txo cov concentration ntawm dawb Mg2+.
7. Template (lub hom phiaj gene) nucleic acid
Tus nqi thiab purification degree ntawm template nucleic acid yog ib qho ntawm cov kev sib txuas tseem ceeb rau kev ua tiav lossis tsis ua tiav ntawm PCR.Cov txheej txheem DNA purification ib txwm siv SDS thiab protease K los zom thiab pov tseg cov qauv.Lub luag haujlwm tseem ceeb ntawm SDS yog: yaj cov lipids thiab cov proteins ntawm cov cell membrane, yog li rhuav tshem cov cell membrane los ntawm kev rhuav tshem cov proteins, thiab dissociate nuclear proteins hauv cell, SDS tuaj yeem ua ke nrog cov proteins thiab precipitate;Protease K tuaj yeem hydrolyze thiab zom cov protein, tshwj xeeb tshaj yog cov histones khi nrog DNA, thiab tom qab ntawd siv cov organic hnyav phenol thiab chloroform los rho cov proteins thiab lwm yam ntawm tes, thiab siv ethanol lossis isopropyl cawv los ua kom cov kua qaub nucleic acid.Cov extracted nucleic acid tuaj yeem siv los ua tus qauv rau PCR cov tshuaj tiv thaiv.Rau cov qauv kuaj pom dav dav, ib txoj hauv kev yooj yim thiab yooj yim siv tau los ua kom cov hlwb, lysate pathogens, zom thiab tshem tawm cov proteins ntawm chromosomes mus rau cov hom phiaj pub dawb, thiab siv ncaj qha rau PCR amplification.RNA template extraction feem ntau siv guanidine isothiocyanate los yog protease K txoj kev los tiv thaiv RNase los ntawm degrading RNA.
8.Mg2+ concentration
Mg2+ muaj qhov cuam tshuam loj rau qhov tshwj xeeb thiab cov txiaj ntsig ntawm PCR amplification.Feem ntau cov tshuaj tiv thaiv PCR, thaum lub concentration ntawm ntau yam dNTP yog 200umol / L, qhov tsim nyog concentration ntawm Mg2 + yog 1.5 ~ 2.0mmol / L.Mg2 + concentration yog siab dhau lawm, cov tshuaj tiv thaiv tshwj xeeb txo qis, tsis muaj qhov tshwj xeeb amplification tshwm sim, cov concentration tsawg dhau yuav txo cov kev ua ntawm Taq DNA polymerase, uas ua rau txo cov tshuaj tiv thaiv cov khoom.
Magnesium ions cuam tshuam rau ntau yam ntawm PCR, xws li DNA polymerase kev ua haujlwm, uas cuam tshuam rau cov qoob loo;Lwm qhov piv txwv yog primer annealing, uas cuam tshuam qhov tshwj xeeb.dNTP thiab template khi rau magnesium ion, txo cov nqi ntawm cov magnesium ion dawb uas yuav tsum tau muaj rau kev ua enzyme.Qhov zoo tshaj plaws magnesium ion concentration sib txawv rau cov sib txawv primer khub thiab templates, tab sis ib tug raug PCR pib concentration nrog 200μM dNTP yog 1.5mM (ceeb toom: Rau real-time quantitative PCR, siv 3 mus rau 5mM magnesium ion tov nrog ib tug fluorescent sojntsuam).Ntau dua cov ntsiab lus ntawm cov dawb magnesium ions nce cov txiaj ntsig, tab sis kuj ua rau kom tsis muaj zog ntxiv thiab txo kev ncaj ncees.Txhawm rau txiav txim siab qhov zoo tshaj plaws, magnesium ion titrations tau ua nyob rau hauv increments ntawm 0.5mM los ntawm 1mM mus rau 3mM.Txhawm rau txo qhov kev cia siab ntawm magnesium ion optimization, Platinum Taq DNA polymerase tuaj yeem siv.Platinum Taq DNA polymerase muaj peev xwm tswj tau kev ua haujlwm ntau dua ntawm magnesium ion ntau dua li Taq DNA polymerase thiab yog li yuav tsum tau ua kom zoo dua.
9. Pcr-txhim kho additives
Optimization ntawm annealing kub, primer tsim, thiab magnesium ion concentration yog txaus rau qhov tshwj xeeb amplification ntawm feem ntau templates;Txawm li cas los xij, qee cov qauv, suav nrog cov ntsiab lus GC siab, xav tau kev ntsuas ntxiv.Cov khoom siv ntxiv uas cuam tshuam rau qhov kub ntawm qhov sib tov ntawm DNA muab lwm txoj hauv kev los txhim kho cov khoom tshwj xeeb thiab tawm los.Tag nrho denaturation ntawm tus qauv yuav tsum tau ua kom tau zoo tshaj plaws.
Tsis tas li ntawd, cov qauv thib ob tiv thaiv primer khi thiab enzyme txuas ntxiv.
PCR additives, suav nrog formamide, DMSO, glycerin, betaine, thiab PCRx Enhancer Solution, txhim kho kev nthuav dav.Lawv cov txheej txheem ua tau yog txhawm rau txo qhov kub thiab txias, yog li pab lub annealing ntawm primers thiab pab DNA polymerase txuas ntxiv los ntawm cov cheeb tsam theem nrab.PCRx Solution muaj lwm yam zoo.Qhov tsawg kawg nkaus magnesium ion optimization yog yuav tsum tau thaum siv nrog Platinum Taq DNA polymerase thiab Platinum Pfx DNA polymerase.Yog li, cov txheej txheem Platinum yog ua ke nrog cov khoom siv ntxiv kom muaj qhov tshwj xeeb thaum txo qhov kev cia siab ntawm peb txoj hauv kev, magnesium ion optimization.Rau cov txiaj ntsig zoo tshaj plaws, cov ntsiab lus ntawm cov tshuaj ntxiv yuav tsum tau ua kom zoo, tshwj xeeb tshaj yog DMSO, formamide, thiab glycerol, uas inhibit Taq DNA polymerase.
10. Kub pib
Kub pib PCR yog ib txoj hauv kev tseem ceeb tshaj plaws los txhim kho PCR tshwj xeeb ntxiv rau kev tsim qauv zoo.Txawm hais tias qhov zoo tshaj plaws elongation kub ntawm Taq DNA polymerase yog 72 ℃, cov polymerase tseem ua haujlwm ntawm chav tsev kub.Yog li, cov khoom uas tsis yog tshwj xeeb yog tsim thaum tuav qhov kub thiab txias qis dua qhov kub ntawm qhov annealing thaum lub sij hawm npaj cov tshuaj tiv thaiv PCR thiab thaum pib ntawm lub voj voog thermal.Thaum tsim, cov khoom tsis tshwj xeeb no tau ua haujlwm zoo.Kub-pib PCR yog tshwj xeeb tshaj yog zoo thaum cov chaw siv rau primer tsim yog txwv los ntawm qhov chaw ntawm noob caj noob ces, xws li site-directed mutations, qhia cloning, los yog kev tsim kho thiab manipulation ntawm noob caj noob ces ntsiab siv rau DNA engineering.
Ib txoj hauv kev los txwv cov kev ua ntawm Taq DNA polymerase yog los npaj cov tshuaj tiv thaiv PCR ntawm cov dej khov thiab muab tso rau hauv lub tshuab PCR preheated.Txoj kev no yog qhov yooj yim thiab pheej yig, tab sis nws tsis ua kom tiav cov haujlwm ntawm cov enzyme thiab yog li ntawd tsis tag tshem tawm cov amplification ntawm cov khoom tsis tshwj xeeb.
Thermal priming ncua DNA synthesis los ntawm inhibiting ib qho tseem ceeb tivthaiv kom txog rau thaum lub PCR apparatus mus txog qhov kub thiab txias denaturation.Feem ntau phau ntawv thermal pib txoj kev, suav nrog ncua sijhawm ntxiv ntawm Taq DNA polymerase, yog cumbersome, tshwj xeeb tshaj yog rau cov ntawv thov siab.Lwm cov txheej txheem thermal priming siv cov ntaub thaiv npog los thaiv cov khoom tseem ceeb, suav nrog magnesium ions lossis enzymes, lossis cais cov khoom siv hauv lub cev, xws li cov qauv thiab cov buffers.Thaum lub sij hawm lub voj voog thermal, ntau yam khoom raug tso tawm thiab sib xyaw ua ke raws li cov quav ciab melts.Zoo li phau ntawv kub pib txoj kev, cov ntaub thaiv npog siv quav ciab txoj kev yog cumbersome thiab nquag mus rau kis kab mob thiab tsis haum rau high throughput daim ntaub ntawv.
Platinum DNA polymerase yog qhov yooj yim thiab ua haujlwm zoo rau kev pib kub pib PCR.Platinum Taq DNA polymerase muaj recombinant Taq DNA polymerase ua ke nrog monoclonal antibody tiv thaiv Taq DNA polymerase.Cov tshuaj tiv thaiv yog tsim los ntawm PCR los tiv thaiv kev ua haujlwm enzyme thaum lub sij hawm ntev kub tuav.Taq DNA polymerase tau tso tawm rau hauv cov tshuaj tiv thaiv thaum lub sij hawm 94 ℃ rwb thaiv tsev ntawm cov kauj ruam denaturation, rov ua kom tag nrho cov haujlwm polymerase.Nyob rau hauv sib piv rau chemically hloov Taq DNA polymerase rau thermal pib, Platinum enzyme tsis xav tau ntev rwb thaiv tsev ntawm 94 ℃ (10 mus rau 15 feeb) kom qhib lub polymerase.Nrog PlatinumTaq DNA polymerase, 90% ntawm Taq DNA polymerase kev ua haujlwm tau rov qab los tom qab 2 feeb ntawm 94 ℃.
11. Nest-PCR
Kev ua tiav ntawm kev nthuav dav siv nested primers tuaj yeem txhim kho qhov tshwj xeeb thiab rhiab heev.Thawj puag ncig yog tus qauv amplification ntawm 15 mus rau 20 cycles.Ib feem me me ntawm cov khoom pib amplification tau diluted 100 mus rau 1000 zaug thiab ntxiv rau qhov thib ob ntawm kev nthuav dav rau 15 mus rau 20 lub voj voog.Xwb, thawj cov khoom lag luam tuaj yeem ua qhov loj me los ntawm gel purification.Ib tug nested primer yog siv nyob rau hauv lub thib ob puag ncig ntawm amplification, uas yuav khi rau lub hom phiaj ib theem zuj zus nyob rau hauv thawj primer.Kev siv nested PCR txo qhov muaj peev xwm ntawm kev nthuav dav ntawm ntau lub hom phiaj vim tias muaj ob peb lub hom phiaj sib txuas ntxiv rau ob pawg ntawm primers.Tib tag nrho cov cycles (30 mus rau 40) nrog tib primers amplified lub site nonspecific.Nested PCR tsub kom qhov rhiab heev ntawm cov hom phiaj tsawg zuj zus (xws li, mrnas tsawg) thiab txhim kho qhov tshwj xeeb ntawm PCRS nyuaj (xws li 5′ RACE).
12. Tus PCR nqis
Descending PCR txhim kho qhov tshwj xeeb los ntawm kev siv nruj nruj rau thawj ob peb lub voj voog ntawm PCR.Lub voj voog pib ntawm qhov ntsuas kub ntawm kwv yees li 5 ℃ siab tshaj qhov kwv yees Tm, tom qab ntawd txhua lub voj voog raug txo los ntawm 1 ℃ mus rau 2 ℃ kom txog rau thaum qhov kub ntawm annealing qis dua Tm 5 ℃.Tsuas yog lub hom phiaj hom phiaj nrog homology siab tshaj plaws yuav raug nthuav tawm.Cov khoom lag luam no txuas ntxiv nthuav dav hauv cov voj voog tom ntej, nthuav tawm cov khoom lag luam amplified nonspecific.Descending PCR yog qhov muaj txiaj ntsig zoo rau txoj hauv kev uas qhov degree ntawm homology ntawm primer thiab lub hom phiaj template tsis paub, xws li AFLP DNA ntiv tes.
Yam khoom siv PCR
PCR Ib qho yooj yim rau kev teeb tsa (nrog Dye)
2 × PCR HeroTMKev sib xyaw ua ke muaj kev ua siab ntev rau PCR inhibitors dua li PCR Mix system, thiab tuaj yeem yooj yim tiv nrog PCR amplification ntawm ntau cov qauv nyuaj.Cov tshuaj tiv thaiv tshwj xeeb thiab kev ua haujlwm siab Taq Hero ua rau PCR cov tshuaj tiv thaiv muaj kev ua kom muaj zog ntau dua, tshwj xeeb thiab rhiab heev.
Ntau dua amplification efficiency
Nws muaj 5'→ 3' DNA polymerase kev ua haujlwm thiab 5'→ 3' exonuclease kev ua, tsis muaj 3'→ 5' exonuclease kev ua.
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Tshwj xeeb-optimized buffer thiab kub-pib Taq enzyme tuaj yeem tiv thaiv tsis muaj qhov tshwj xeeb amplification thiab primer dimer tsim
Siab rhiab heev - tuaj yeem ntes cov ntawv luam tsawg ntawm cov qauv
RT-PCR Ib qho yooj yim thiab yooj yim rau kuv (Ib kauj ruam)
Cov khoom siv siv ib qho tshwj xeeb Foregene thim rov qab reagent thiab Foregene HotStar Taq DNA Polymerase ua ke nrog cov tshuaj tiv thaiv tshwj xeeb txhawm rau txhim kho kev ua kom zoo dua qub thiab qhov tshwj xeeb ntawm cov tshuaj tiv thaiv.
Post lub sij hawm: May-09-2023
