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

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Answer

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: E1083

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165bc95226f930b45454eb916c9ac3af_FAQ

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: E1084

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6c32f5d0085dc5fa45143988d1afd068_FAQ

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

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Answer

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: E1086

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a6af3039cea44c35076430e42b3a79fc_FAQ

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: E1088

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fb03555d02e802cc19fdb5f3b7d11622_FAQ

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: E1098

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e9f01c60c3a77427d063148da7cdccda_FAQ

What is the expected half life of AmpliTaq® DNA Polymerase at 95 degrees C?

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The half-life of AmpliTaq® DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq® DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.

Answer Id: E1139

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11f0398279d14cfe21e3a1adc1654dae_FAQ

Does the fidelity of AmpliTaq® DNA Polymerase change in the presence of base analogs?

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Answer

The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq® DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.

Answer Id: E1335

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db9c1cc986291525e9399346d1d5b8ae_FAQ

Does AmpliTaq® Gold DNA Polymerase contain exonuclease (proofreading) activity?

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Answer

No, AmpliTaq® Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.

Answer Id: E1338

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1d2303aa8dfec1db71c18c18581d3e1c_FAQ

How does AmpliTaq Gold® DNA Polymerase differ from AmpliTaq® DNA Polymerase?

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Answer

AmpliTaq Gold® DNA Polymerase is a modified form of AmpliTaq® DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold® DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp® 10X PCR Buffer I and/or GeneAmp® 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp® 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Answer Id: E1339

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4a68e0b4f83700f53c291e81b82625bc_FAQ

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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Answer

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

Answer Id: E1408

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cd618c8ed9d56976a4230f1153eec906_FAQ

Do Elongase® and Platinum® Taq High Fidelity enzymes leave a 3'-A overhang on the PCR product for subsequent cloning into a TOPO® TA Cloning® or original TA vectors? What about Platinum® Pfx polymerase?

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Answer

Elongase® and Platinum® Taq High Fidelity polymerase mixes do leave 3' A overhangs on a portion of the PCR products, however, the cloning efficiency is greatly reduced from that obtained with Taq polymerase alone. Platinum® Pfx polymerase does not leave 3' A overhangs. Therefore, with all proofreading enzymes or enzyme mixes that contain proofreading polymerases, we recommend that you treat the PCR product with Taq at the end of the PCR reaction, prior to TA cloning. To do this, add 1 U of Taq to a 50 μL reaction and incubate at 68-72 degrees C for 15 min. Phenol extract and ethanol precipitate the product before TA cloning.

Additional notes: The cloning efficiency decreases with increasing size of PCR products. For larger PCR fragments, we recommend that you gel-purify the PCR product and screen several clones. PCR primers should be designed with a 5' G, since Taq leaves a 3' A overhang preferentially on DNA ending in C.
Reference: Hu (1993) DNA and Cell Biology 12:763.
TA Cloning reference: Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., and Smith, L.M. (1991) BioTechnology 9, 657.

Answer Id: E3064

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5b5f8fa48225c7ffe5c7bfcd188cef48_FAQ

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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Answer

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.

Answer Id: E4024

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815dae5afa8bbcc366587e4d3d919944_FAQ

How does TA Cloning® work?

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Answer

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: E4061

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fb53c1ece88718be1d4da1509411423b_FAQ

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

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Answer

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: E4062

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340dfd4c1b12d5bd85bdb219d563375d_FAQ

Can you compare the DNA polymerases you offer by fidelity, maximum amplicon length, and 3’ A-overhang?

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Answer

Please see the comparison below on the following criteria:
Enzyme, Relative Fidelity, Amplicon Length, and 3’ Overhang (+/-)
Taq, 1, <5 kb, +
Platinum® Taq, 1, <10 kb, +
AccuPrime™ Taq, 2, <5 kb, +
Platinum® Pfx, 26, <12 kb, -
AccuPrime™ Pfx, 26, <12 kb, -
Pfx50™, 50, <4 kb, -
Platinum® Taq HiFi, 6, <20 kb, +/-
AccuPrime™ Taq HiFi, 9 <20 kb, +/-
AmpliTaq®, 1, <5 kb, +
AmpliTaq Gold®, 1, <5 kb, +
AmpliTaq Gold® 360, 1, <5 kb, +

Taq error rate: 1 x 10-4 to 2 x 10-5 base/duplication

Answer Id: E7263

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2cbb9a8b371d1a76df284db2645c5d25_FAQ