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Successful PCR Conditions Uspješna PCR uvjeti

Parameters for Successful PCR Parametri za uspješno PCR

Are you having problems of achieving a successful PCR?  Many factors can affect your outcome of your PCR such as: Metal Ion Cofactors, Substrate and Substrate Analogs, Buffers and Salts and Cosolvents.  Also Thermal Cycling Considerations:  PCR Vessels, Temperature and Time Optimization, PCR Amplification Cycles, Enzyme/Target and Hot Start. Jeste li problema postizanja uspješnog PCR? Mnogi faktori mogu utjecati na ishod vaših PCR kao što su: Metal Ion Cofactors, podloga i podloga Analogs, Buffers i soli i Cosolvents. Također Termalni Biciklizam razmatranja: PCR posuđe, temperature i vremena za optimizaciju, PCR amplifikacija Cycles, enzim / target i Hot Start.

Metal Ion Cofactors and PCR Metal Ion Cofactors i PCR

An essential cofactor for the DNA polymerase in PCR is Magnesium chloride.  Its concentration must be optimized for every primer:template system. Bitan cofactor za DNA polymerase u PCR je Magnezij klorid. Njezin koncentracija mora biti optimiziran za svaki udžbenik: template sustav. Many components of the reaction bind magnesium ion, including primers, template, PCR products and dNTPs. Mnoge komponente reakcija svezati ion magnezija, uključujući i početnice, predložak, PCR proizvoda i dNTPs. The main 1:1 binding agent for magnesium ion is the high concentration of dNTPs in the reaction. Glavni 1:1 binding agent za magnezij ion je visoka koncentracija dNTPs u reakciji. Because it is necessary for free magnesium ion to serve as an enzyme cofactor in PCR, the total magnesium ion concentration must exceed the total dNTP concentration. Zato što je potrebno za besplatni ion magnezija da služi kao enzim cofactor in PCR, ukupni magnezij ion concentration mora premašiti ukupne dNTP koncentracija. Typically, to start the optimization process, 1.5 mM magnesium chloride is added to PCR in the presence of 0.8 mM total dNTPs. Tipično, kako bi se pokrenuo proces optimizacije, 1,5 mm, magnezij klorid je dodao da PCR u prisutnosti 0,8 mm ukupna dNTPs. This leaves about 0.7 mM free magnesium for the DNA polymerase. Ovo ostavlja oko 0,7 mm slobodnog magnezija za DNA polymerase. In general, magnesium ion should be varied in a concentration series from 1.5–4.0 mM in 0.5 mM steps. Općenito, magnezij ion bi trebao biti raznolika u koncentraciji iz serije 1.5-4.0 mm u 0,5 mm korake.

Substrates and Substrate Analogs for PCR Podlogama i podloga Analogs za PCR

The DNA polymerases incorporate very efficiently dNTPs.  They also can incorporate modified substrates, when they are used as supplemental components in PCR. The DNA polymerases inkorporirati vrlo efikasno dNTPs. Oni također mogu uključivati izmijenjeni podlogama, kada se koriste kao dodatne komponente u PCR. Examples of substrates used for DNA polymerase are: Digoxigenin-dUTP, biotin-11-dUTP, dUTP, c7deaza-dGTP, and fluorescently labeled dNTPs. Primjeri podlogama koristiti za DNA polymerase su: Digoxigenin-dUTP, biotin-11-dUTP, dUTP, c7deaza-dGTP, i fluorescently etiketom dNTPs. For conventional PCR, the concentration of dNTPs remains balanced in equimolar ratios, eg, 200 μM each dNTP. Za konvencionalne PCR, koncentracija dNTPs ostaje uravnotežena u equimolar Pokazivači, npr., 200 μ m dNTP. Also note that, deviations from these standard recommendations may be beneficial in certain amplications. Također, imajte na umu da, odstupanja od ovih standardnih preporuka svibanj biti koristan u nekim amplications. For example, when random mutagenesis of a specific target is desired, unbalanced dNTP concentrations promote a higher degree of misincorporations by the DNA polymerase. Na primjer, kada slučajnih mutagenesis of određenu ciljnu je željeni, neuravnotežen dNTP koncentracije promiču viši stupanj misincorporations po DNA polymerase.

Buffers and Salts for PCR Buffers i soli za PCR

Depending on the DNA polymerase used the optimal PCR buffer concentration, salt concentration, and pH should be picked accordingly to the DNA polymerase. Ovisno o DNA polymerase koriste optimalno PCR tampon koncentracija, koncentracija soli, a pH bi trebao biti izabrane u skladu s DNA polymerase. The PCR buffer for Taq DNA polymerase consists of 50 mM KCl and 10 mM Tris-HCl, pH 8.3, at room temperature. The PCR tampon za Taq DNA polymerase se sastoji od 50 mm i 10 mm KCl-Tris HCL, pH 8,3, na sobnoj temperaturi. This buffer provides the ionic strength and buffering capacity needed during the reaction. Ova tampon pruža ionic snagu i razdvajanje kapacitet potreban da bi se tijekom reakcije. It is important to note that the salt concentration affects the Tm of the primer:template duplex, and hence the annealing temperature. Važno je imati na umu da je koncentracija soli utječe na Tm od prvog: predložak duplex, i otud žarenje temperatura.

Cosolvents

Different PCR Cosolvents are used to increase the yield, efficacy, and specificity of PCR amplifications. Različitih PCR Cosolvents se koriste za povećanje prinosa, djelotvornost i specifičnost od PCR amplifications. These cosolvents can be advantageous in some amplifications, and disadvantageous in other amplifications. Ove cosolvents mogu biti korisni u nekim amplifications, i disadvantageous u drugim amplifications. It is impossible to predict which additive will be useful for each primer:template duplex and therefore the cosolvent must be empirically tested for each combination. To je nemoguće predvidjeti što će biti s dodanim koristan za svaki udžbenik: duplex predloška i zato su cosolvent mora biti ispitan empirički za svaku kombinaciju.

Thermal Cycling Considerations Termalni Biciklizam razmatranja

PCR Vessels PCR posuda

PCR must be performed in vessels that are compatible with low amounts of enzyme and nucleic acids and that have good thermal transfer characteristics. PCR mora biti izvedena u posude koje su kompatibilne s niskim količinama enzim i nukleinske kiseline i da imaju dobre termalne karakteristike prijenosa. Usually, polypropylene is used for PCR vessels and conventional, thick-walled microcentrifuge tubes are chosen for many thermal cycler systems. Obično, polipropilen se koristi za PCR posuđe i konvencionalna, debelih stijenki microcentrifuge-cijevi su izabrani za brojne termalne cycler sustava. PCR is most often performed at a 10–100 μL reaction scale and requires the prevention of the evaporation/condensation processes in the closed reaction tube during thermal cycling. PCR se najčešće izvodi na 10-100 μ L skale i zahtijeva reakciju na prevenciji i isparavanju / kondenzacija procesi u zatvorenim reakcija tube tijekom termalne biciklizam. A mineral oil overlay or wax layer serves this purpose. A mineralna ulja overlay ili vosak sloj služi tu svrhu. More recently, 0.2-mL thin-walled vessels have been optimized for the PCR process and oil-free thermal cyclers have been designed that use a heated cover over the tubes held within the sample block. Više nedavno, 0.2-mL tankostijene plovila su optimizirane za PCR postupak i ulje-free termalne cyclers su dizajnirani da koriste grijanom pokrivaju preko cijevi održan u uzorku blok.

Temperature and Cycle Time Optimization Temperatura i ciklus Vrijeme Optimizacija

It is essential that the reaction mixtures reach the denaturation, annealing, and extension temperatures in each thermal cycle. To je bitno da je reakcija mješavina doći do denaturation, žarenje, temperatura i proširenje u svakom ciklusu termalnih. If insufficient hold time is specified at any temperature, the temperature of the sample will not be equilibrated with that of the sample block. Ako je nedovoljno vrijeme čekanja je naveden u bilo kojem temperatura, temperatura u uzorku neće biti equilibrated s koje je uzorak blok. Some thermal cycler designs time the hold interval based on the block temperature, whereas others base the hold time on predicted sample temperature. Neki termo-cycler dizajne vrijeme na čekanju interval na temelju blok temperatura, dok se drugi osnovni je vrijeme čekanja na predviđeni uzorak temperatura. If a conventional thick-walled tube used in a cycler controlled by block temperature, a 60-s hold time is sufficient for equilibration. Ako konvencionalne debelih stijenki-cijev koristi u cycler kontrolira blok temperature, 60-e vrijeme čekanja je dovoljno za equilibration. Extra time may be recommended at the (72°C) extension step for longer PCR products. Extra vrijeme svibanj biti preporučeno na (72 ° C) ekstenziju za korak više PCR products. Using a thin-walled 0.2-mL tube in a cycler controlled by predicted sample temperature, only 15 s is required. Korištenje tankostijene 0,2 mL tube-u cycler kontrolira predviđeni uzorak temperatura, a samo 15 je obavezna. To use existing protocols or to development protocols for use at multiple laboratories, it is very important to choose hold times according to the cycler design and tube wall thickness. Da biste koristili postojeće protokole ili na razvoju protokola za korištenje na više laboratorijima, vrlo je važno odabrati čekanje puta prema cycler dizajn i tube zid debljine.

PCR Amplification Cycle Number PCR amplifikacija Ciklus Broj

The number of PCR amplification cycles should be optimized with respect to the starting concentration of the target DNA. Broj PCR amplifikacija ciklusa bi trebao biti optimiziran s obzirom na početnu koncentraciju ciljanu DNA. It is recommended that, from 40– 45 cycles to amplify 50 target molecules, and 25–30 cycles to amplify 3 × 105 molecules to the same concentration. Preporuča se da, od 40 - 45 ciklusa u 50 povećati ciljanu molekula, i ciklusima 25-30 povećati na 3 × 105 molekula u istoj koncentraciji. This non-proportionality is caused by a so-called plateau effect, in which a decrease in the exponential rate of product accumulation occurs in late stages of a PCR. To ne-proporcionalnosti je uzrokovana tzv visoravni efekt, u kojoj je smanjenje u eksponencijalna stopa akumulacije proizvod pojavljuje u kasnim fazama od PCR. This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs); end-product inhibition (pyrophosphate formation); competition for reactants by non-specific products; or competition for primer binding by reannealing of concentrated (10 nM) product. Ovaj svibanj biti uzrokovan degradacija reactants (dNTPs, enzim); reactant osiromašeni (početnice, dNTPs); end-product inhibicija (pyrophosphate formation); natječaj za reactants by non-određenim proizvodima, ili natjecanje za upaljač obvezujuća by reannealing, koncentrirana ( 10 Nm) proizvod. It is usually advisable to run the minimum number of cycles needed to see the desired specific product, because unwanted nonspecific products will interfere if the number of cycles is excessive. To je obično poželjno izvoditi minimalnog broja ciklusa je potrebno da biste vidjeli željeni određenog proizvoda, jer će neželjeni nespecifična proizvodi uplitati se, ako je broj ciklusa je pretjeranim.

Enzyme / Target Enzim / target

In a standard aliquot of Taq DNA polymerase used for a 100-μL reaction, there are about 1010 molecules. U standardnom aliquot of Taq DNA polymerase korišten za 100 - μ L reakcija, ima oko 1010 molekula. Each PCR sample should be evaluated for the number of target copies it contains or may contain. Svaki PCR uzorak treba biti ocijenjen za broj ciljanih kopija sadrži ili svibanj sadržavati. For example, 1 ng of lambda DNA contains 1.8 × 107 copies. Na primjer, od 1 of lambda DNA sadrži 1,8 × 107 primjeraka. For low-input copy number PCR, the enzyme becomes limiting and it may be necessary to give the extension process incrementally more time. Za low-input primjerak broj PCR, enzim postaje ograničavajući i svibanj biti potrebno dati ekstenziju proces incrementally više vremena. Thermal cyclers can reliably perform this automatic segment extension procedure in order to maximize PCR yield. Termalni cyclers može pouzdano obavljati ovaj segment ekstenziju automatski postupak kako bi se povećala PCR prinosa.

Hot Start Conditions Hot Start odredbe

All of the above optimizations also apply to a PCR that is designed, from the beginning, with a hot start method. Sve gore navedene optimizacije također se primjenjuju na PCR koji je projektiran, od početka, sa hot start metoda. Often, a hot start can be incorporated successfully into a previously optimized PCR without changing the reaction conditions. Često, hot start može biti uspješno uključene su u prethodno optimized PCR bez mijenjanja reakcija uvjetima. However, it usually pays to reoptimize after adding a hot start. Međutim, obično se plaća na reoptimize nakon dodavanja hot start. Optimization is often a balance between producing as much product as possible and overproducing nonspecific, background amplifications. Optimizacija je često ravnotežu između proizvodnje proizvoda kao što je više moguće i overproducing nespecifična, background amplifications. Because hot start greatly reduces background amplifications, the upper restraints are raised on conditions such as enzyme concentration, cycle number, and metal ion cofactor concentration. Zbog hot start uvelike smanjuje pozadini amplifications, gornji restraints su podigli na uvjete kao što je enzim koncentracija, broj ciklusa, a cofactor metal ion concentration. Sensitive PCRs that have been highly tuned without a hot start may fail when a hot start is added. Osjetljive PCRs da su vrlo usklađeno, bez hot start svibanj fail kada je dodano hot start. This can be caused by slight delays in early cycles caused by mixing or enzyme activation. To mogu biti uzrokovane neznatno odgode u ranim ciklusa uzrokovana miješanje ili enzim za aktivaciju. The PCR usually can be restored, often with substantial increase in specific product, by simply increasing limiting parameters or reagents. The PCR obično može biti obnovljena, često uz znatno povećanje u određenim proizvodom, tako jednostavno ograničavati povećanje parametara ili Reagensi. In addition, there are optimizations specific to each hot start method. Osim toga, tu su i poboljšanja su specifične za svaku hot start metoda. Mixing or enzyme activation can be affected by PCR volume, buffer composition and pH, cosolvents, cycling conditions, and so on. Miješanje ili enzim za aktivaciju mogu biti pogođeni PCR volume, tampon sastav i pH, cosolvents, biciklizam uvjete, i tako dalje. The specific product’s literature, often a product insert, should be consulted for information on these considerations. Specifičan proizvod's literature, često proizvod ubacite, trebali bi se konzultirati za informacije o ovim razmisljanjima.