What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

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Successful amplification of long PCR targets is dependent variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. Routine amplification of targets up to 5 kb has been successful with the use of AmpliTaq® Polymerase and AmpliTaq Gold® DNA Polymerase under standard PCR buffer conditions. However, for targets ranging from 5 kb up to 40 kb, rTth DNA Polymerase, XL works optimally. This enzyme and its optimized buffer promote not only efficient DNA synthesis, but also allow for correction of nucleotide misincorporations that might otherwise prematurely terminate synthesis. Please refer to the GeneAmp® XL PCR Kit Product Insert for more information.

Answer Id: 1083

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When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?

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The concentration of dNTPs in a standard PCR amplification is 200 μM each, for a total of 800 μM. This total dNTP amount corresponds to 39 μg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 μg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.

Answer Id: 1084

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How much AmpliTaq® DNA Polymerase is used in a PCR amplification?

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Most PCR amplifications use 2.5 units of AmpliTaq® DNA polymerase per 100 μL reaction. A 25 μL reaction would use about 0.6 units. However, the optimal unit concentration per reaction should be empirically determined, and often the less is better rule applies. Using too much AmpliTaq® may result in non-specific amplification.

Answer Id: 1086

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How much MgCl2 should be added to the PCR amplification when using AmpliTaq® DNA Polymerase or AmpliTaq Gold® DNA Polymerase?

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The standard starting point is a final concentration of 1.5 mM magnesium ion. Since each molecule of dNTP (total 0.8 mM per reaction at 200 μM each) binds a magnesium ion, 0.8 mM magnesium ions are unavailable for AmpliTaq® DNA Polymerase to use; hence, 0.7 mM free magnesium ions will be available as a cofactor for Taq's polymerization activity. It is important to note that there are other substrates in PCR amplifications that can also bind free magnesium (such as primers and template) therefore, the magnesium ion concentration should be titrated in order to find the optimum concentration for each reaction.

Answer Id: 1088

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What is "Hot-start" PCR?

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Hot-start is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.

In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and specific amplification occurs.

AmpliTaq Gold® DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold® DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.

Answer Id: 1098

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Is there anything to prevent AmpliTaq Gold® DNA polymerase from extending from the 3’ end of a TaqMan® probe in a 5’ nuclease assay?

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Yes. There is a phosphate group on the 3' end of all TaqMan® probes that prevents such extension.

Answer Id: 1408

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What are the melting temperatures for the M13 Forward (-20) and M13 Reverse primers in the TOPO® Cloning and Zero Blunt® Kits?

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Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.

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How does TA Cloning® work?

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Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning® kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

Answer Id: 4061

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Can I use a DNA polymerase mixture containing both Taq polymerase and a proofreading polymerase for TA Cloning®?

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If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.

Some examples of Taq blends that are compatible with TOPO® TA Cloning® are Platinum® Taq DNA Polymerase High Fidelity and AccuPrime™ Taq DNA Polymerase High Fidelity.

Answer Id: 4062

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