Kuv ntseeg tias txhua tus neeg yuav ib txwm ntsib cov teeb meem zoo li no lossis cov teeb meem zoo li no thaum ua cov tshuaj tiv thaiv PCR, tab sis feem ntau ntawm lawv tuaj yeem muab faib ua ob qhov teeb meem loj:

Kev nthuav dav me me ntawm cov qauv qauv (amplification);
Ntau dhau tsis yog hom phiaj gene amplification.
Kev siv cov tshuaj ntxiv yog ib qho ntawm cov tswv yim zoo los daws cov teeb meem no.Feem ntau lub luag hauj lwm ntawm additives muaj ob yam:
theem nrab qauvntawm cov noob (cov qauv thib ob);
Txo cov priming tsis tshwj xeeb.
Niaj hnub no, tus editor yuav qhia luv luv rau koj txog cov tshuaj ntxiv hauv PCR cov tshuaj tiv thaiv thiab lawv cov haujlwm.
Additives uas txo cov qauv theem nrab
sulfoxide(DMSO)
cov qauv kuajnrog siab GC cov ntsiab lus.Txawm li cas los xij, DMSO kuj tseem txo qis Taq polymerase kev ua haujlwm.Yog li ntawd, txhua tus neeg yuav tsum sib npaug ntawm cov qauv siv tau thiab cov haujlwm ntawm cov polymerase.Tus editor qhia tias koj tuaj yeem sim ntau qhov sib txawv ntawm DSMO, xws li los ntawm 2% mus rau 10%, kom pom qhov concentration uas haum rau koj qhov kev sim.
Tsis-ionic detergents
Non-ionic detergents, xws li 0.1-1% Triton X-100, Tween 20 lossis NP-40, feem ntau txo cov qauv DNA thib ob.Txawm hais tias qhov no tuaj yeem ua rau kom muaj kev nthuav dav ntawm cov qauv gene, nws tseem yuav ua rau muaj teeb meem ntawm qhov tsis tshwj xeeb amplification.Yog li, cov additives no ua haujlwm zoo rau cov txiaj ntsig PCR qis uas tsis muaj cov khib nyiab, tab sis tsis zoo rau cov tshuaj tiv thaiv PRC tsis zoo.Lwm qhov txiaj ntsig ntawm cov tshuaj ntxuav tes uas tsis yog ionic yog txo cov kab mob SDS.Feem ntau thaum lub sij hawm DNA extraction txheej txheem, SDS yuav raug coj mus rau PCR kauj ruam, uas zoo heev inhibits cov kev ua ntawm polymerase.Yog li ntawd, ntxiv 0.5% Tween-20 los yog Tween-40 rau cov tshuaj tiv thaiv tuaj yeem cuam tshuam qhov tsis zoo ntawm SDS.
Betaine_
Betaine tuaj yeem txhim kho DNA amplification los ntawm kev txo qis cov qauv tsim thiab feem ntau yog "tsis paub" ntxiv rau cov khoom siv PCR coj mus muag.Yog tias koj xav siv betaine, koj yuav tsum muab betaine lossis betaine mono-hydrate (Betaine lossis Betaine mono-hydrate), tab sis tsis yog betaine hydrochloride (Betaine HCl), kho kom haum rau qhov kawg ntawm 1-1.7M.Betaine kuj tseem tuaj yeem pab txhim kho qhov tshwj xeeb vim tias nws tshem tawm lub hauv paus khub sib xyaw ua ke ntawm DNA melting / DNA denaturation.
Additives los txo qhov tsis yog tshwj xeeb priming
Formamide
Formamide yog ib qho khoom siv organic PCR ntxiv.Nws tuaj yeem ua ke nrog cov zawj loj thiab me zawj hauv DNA, yog li txo qhov kev ruaj ntseg ntawm tus tswv DNA ob lub helix thiab txo qhov kub ntawm DNA.Qhov concentration ntawm formamide siv hauv PCR kev sim feem ntau yog 1% -5%.
Tetramethylammonium chloride( TMAC)
Tetramethylammonium chloride tuaj yeem ua rau muaj qhov tshwj xeeb ntawm hybridization (hybridization tshwj xeeb) thiab ua rau kom qhov kub thiab txias ntawm DNA.Yog li, TMAC tuaj yeem tshem tawm tsis yog tshwj xeeb priming thiab txo cov misbinding ntawm DNA thiab RNA.Yog koj sivdegenerate cov primersHauv cov tshuaj tiv thaiv PCR, nco ntsoov ntxiv TMAC, uas feem ntau siv ntawm qhov concentration ntawm 15-100mM.
Lwm yam Additives
Ntxiv rau ob pawg ntawm cov khoom ntxiv uas tau hais los saum toj no, muaj ntau cov tshuaj ntxiv hauv PCR cov tshuaj tiv thaiv, txawm tias lawv muaj cov haujlwm sib txawv, lawv kuj tseem ceeb heev.
Magnesium ion
Magnesium ion yog ib qho tseem ceeb ntawm cofactor (cofactor) ntawm polymerase, uas yog hais tias, tsis muaj magnesium ion, polymerase tsis ua haujlwm.Txawm li cas los xij, magnesium ions ntau dhau tuaj yeem cuam tshuam rau kev ua haujlwm ntawm polymerase.Qhov concentration ntawm magnesium ions hauv txhua qhov tshuaj tiv thaiv PCR yuav txawv.Chelating agents (xws li EDTA los yog citrate), qhov concentration ntawm dNTPs thiab cov proteins tag nrho cuam tshuam rau cov concentration ntawm magnesium ions.Yog li, yog tias koj muaj teeb meem nrog koj qhov kev sim PCR, koj tuaj yeem sim hloov qhov sib txawv magnesium ion concentrations, piv txwv li, los ntawm 1.0 mus rau 4.0mM, nrog rau lub caij nyoog ntawm 0.5-1mM nyob nruab nrab.
Nws yog ib qho tsim nyog sau cia tias ntau lub voj voog khov-thaw tuaj yeem ua rau kom muaj zog stratification ntawm magnesium chloride tov.Yog li ntawd, koj yuav tsum yaj nws tag nrho ua ntej siv, thiab sib tov zoo ua ntej siv nws.
Bovine serum albumin(Bovine albumin, BSA)
Hauv kev sim tshuaj molecular chemistry, bovine serum albumin yog ib qho tshuaj ntxiv, tshwj xeeb tshaj yog nyob rau hauv kev txwv enzyme digestion thiab PCR thwmsim.Hauv PCR cov tshuaj tiv thaiv, BSA pab txo cov kab mob xws li phenolic compounds.Thiab nws kuj tau hais tias nws tuaj yeem txo qhov adhesion ntawm reactants rau phab ntsa ntawm lub raj kuaj.Hauv cov tshuaj tiv thaiv PCR, feem ntau qhov concentration ntawm BSA ntxiv tuaj yeem ncav cuag 0.8 mg / ml.
 
Yam khoom:
PCR Hero(nrog dye)
PCR Hero