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View additional product information for Platinum™ Taq DNA Polymerase - FAQs (10966026, 10966018, 10966083, 10966034)
53 product FAQs found
The 10X PCR buffer for Platinum Taq is not available as a stand-alone item. It is only supplied as part of the enzyme kit.
Oligos should be run on a polyacrylamide gel containing 7 M urea and loaded with a 50% formamide solution to avoid compressions and secondary structures. Oligos of the same length and different compositions can electrophorese differently. dC's migrate fastest, followed by dA's, dT's, and then dG's. Oligos containing N's tend to run as a blurry band and generally have a problem with secondary structure.
Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.
The drying method dries the primer in a thin layer along the sidewalls of the tube instead of the bottom, therefore a pellet is not always visible and should still be ready to use.
If the oligo was overheated, it will appear as a “ball”-shaped pellet attached to the bottom of the tube. This should not affect the quality of the oligo, and the oligo should be readily soluble in water.
If an oligo appears green in color, this is most likely due to ink falling into the tube. The oligo should still be fully functional. The color can be removed by doing an ethanol precipitation.
Most of the time the color should not affect PCR or any other experimental application since typically it is caused by the iodine used in the synthesis. There are some exceptions, however. Brown oligos can also be caused by the primer being overdried, and if this is the case, the primer may not work.
If detritylation occurs inappropriately and/or if the synthesizer has an error and delivers the wrong base, an extra inserted base can occur in your primer. Please contact techsupport@thermofisher.com for assistance.
There are two possibilities that could occur in any round of extension when creating your primer:
1.The added base is not detritylated correctly, missing one base addition but allowing possible extension in the next round.
2.The trityl group was removed, but not coupled or capped correctly before addition of the next base, allowing the chain to continue.
Better purification of the oligos is recommended to provide you with full-length oligo sequence. Adding restriction sites adds on 10 or more bases to the basic 20-25-mer, making primers longer than 30 bases with a relatively low percentage of full-length sequences after desalting. Additionally, failure sequences occur at the 5' end of the sequence as oligos are generated from 3' to 5' end. Therefore, restriction sites introduced at the 5' end of primers can be compromised, resulting in missing bases.
The oligo may not have been fully solubilized. After addition of TE buffer, make sure the oligo was vortexed for a full 30 seconds and/or pipette up and down more than 10 times. Primers may be present along the sides of the tubs, so when resuspending the oligo, the sides of the tubes should be “rinsed” too.
The scale that is ordered refers to the starting synthesis scale, or amount of starting material used to create your oligo. Based on purification and efficiency, you will receive less than the starting synthesis scale. However, we do have a minimum yield guarantee based on the starting synthesis scale which can be found here: https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html.
This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction. Try heating to 65 degrees C and putting sample on ice before loading.
Please see the following possibilities and suggestions we have:
-Primer design: try longer primers to avoid binding at alternative sites, avoid 3 consecutive G or C nucleotides at the 3' end.
-Annealing temperature: increase annealing temperature to increase specificity.
-Mg2+ concentration: try a lower concentration.
-DNA contamination: use aerosol tips and separate work area to avoid contamination, use UNG/UDG technique to prevent carryover.
Here are some reasons why your PCR experiment may be failing:
-NaCl at 50 mM will inhibit the enzyme.
-Too much KCl in the reaction. Do not exceed 50 mM.
-Incorrect annealing temperature was used.
-Incomplete denaturation (time and temperature must be long and high enough).
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR].
-10% DMSO partially inhibits Taq.
-Hemin (in blood samples) inhibits Taq.
-Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR [Lee (1995) BioTechniques 18:225].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the amount of Taq, and/or increase the volume of the PCR to dilute out them inhibitor.
Please see our suggestions below to increase yield:
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR. [Lee (1995) BioTechniques 18:225].
-Not enough enzyme was used.
-Denaturation/extension temperature was too high and enzyme died prematurely.
-Too much DMSO (>10%).
-Incorrect annealing temperature: run a series of reactions using different annealing temperatures, starting 5 degrees below the calculated Tm.
-Too few cycles.
-Insufficient or too much Mg2+.
-Poorly designed primers: double check primer sequence against template sequence, primers should have similar melting temperatures, avoid complementary sequences at the 3' end of primers.
-Carryover inhibitors (e.g., blood, serum).
-Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.
-Not a long enough extension time was used depending on the size of product being amplified.
-Use of super-irradiated (treated with >2500 mj/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6-8% DMSO or 5% DMSO with 1.2-1.8 M betaine can be used to amplify GC-rich templates [Baskaran (1996) Genome Res 6:633].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin, etc.). Add BSA to the PCR (~160-600 µg/mL), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor. The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, fatty acid-free, alcohol-precipitated BSA, or Fraction V BSA all should be effective.
Please see some reasons below for seeing smearing:
-The enzyme, primer, Mg2+, and/or dNTP concentration was too high.
-The annealing temperature was too low for the primers being used.
-Too many cycles were used.
-The annealing and extension times were too long.
-Bad or old primers.
-Too much template was used initially, try to start with 104-106 molecules
-Consider using additives or PCR Optimizer Kit (Cat. No. K122001), especially if you feel strongly that the primers should work/have worked before and are using Taq.
No, we do not guarantee 50/50 of mixed bases. If a mix of GC bases is requested, for example, the synthesizer would deliver half the normal amount of G and half the normal amount of C. Coupling efficiency is not taken into account. Therefore, it is possible that a mix, such as 30/70, will be delivered.
For 25, 50, and 200 nmol desalted and cartridge-purified DNA oligos, there is 100% A260 analysis. Random samples of 25% of the oligos produced are tested by either capillary electrophoresis or mass spectrometry. DNA oligos that are desalted and ordered at 25 and 50 nmol scales also have 100% real-time digital trityl monitoring during analysis. Desalted DNA oligos ordered at 1 and 10 µmols, DNA oligos at any scale that are purified by HPLC and PAGE, the majority of the DNA oligos with 3' and/or 5' modifications, and RNA oligos have 100% A260 analysis and capillary electrophoresis or mass spectrometry.
The plate orders must contain an average of 24 or more oligos per plate for 96-well plates or 192 or more oligos per plate for 384-well plates across the entire order.
Tm values are not absolute - they are an approximation of the melting temperature range which exists. A thermal profile for a given oligo shows a 10-15 degree range of melting depending on the amount of salt but also on the base composition and concentration of primer in the reaction which are not precisely defined. One should not rely solely on the given Tm value as the only one that will work. Tm is the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule. The Tm is necessary to establish an annealing temperature for PCR. Reasonable annealing temperatures range from 55 degrees C to 70 degrees C. Annealing temperatures are generally about 5 degrees C below the Tm of the primers. Since most formulas provide an estimated Tm value, the annealing temperature is only a starting point. Specificity for PCR can be increased by analyzing several reactions with increasingly higher annealing temperatures.
As oligos increase in length, the column purification is less effective in separating the failure oligos from the correct products. PAGE purification would be the method of choice in this case.
Value Oligos are the most cost-effective and fastest way to order oligos. They are available for 5-40-mers, at a 25 or 50 nanomole scale, with a range of purification options to suit your needs, and are eligible for next-day delivery. The cost is calculated per oligo as opposed to per base. Value Oligos are not available with modifications. Value Oligos undergo the same QC standards as our standard oligos with the same manufacturing process.
A common equation used to calculate primer Tm is as follows: Tm (in degrees C) = 2 (A+ T) + 4 (G + C)
Please take a look at this list (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-modification-options.html) of standard modification options that we offer. If you do not see the modification option you would like, please email our Technical Support team at techsupport@thermofisher.com to see if we can accommodate your request.
The percentage of full-length oligonucleotide depends on the coupling efficiency of the chemical synthesis. The average efficiency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula: 0.99n-1. Therefore, 79% of the oligonucleotide molecules in the tube are 25-bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purification.
Coupling efficiency is important as the effects are cumulative during DNA synthesis. The numbers below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product available following synthesis of different length oligos. Even with a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can mean 15% more of the DNA present following synthesis is full-length product.
Number of bases added, 99% coupling full-length, Failures, 98% coupling full-length, Failures:
- 1, 99, 1, 98, 2
- 2, 98.01, 1.99, 96.04, 2.96
- 3,97.03, 2.97, 94.12, 5.88
- 10, 90.44, 9.56, 81.71, 18.29
- 20, 81.79, 18.21, 66.76, 33.24
- 30, 73.79, 26.03, 54.55, 63.58
- 50, 60.5, 39.5, 36.42, 63.58
- 95, 38.49, 61.51, 14.67, 85.33
The scale of synthesis is the starting point for synthesis, not the guaranteed final amount. We guarantee the total yield of oligonucleotide as a minimum number of OD units. Use this link (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html) for the minimum yield guarantees we offer for our oligos.
Yes. OligoPerfect Designer can be used to design primers for sequencing, cloning, or detection.
These guidelines may be useful as you design your PCR primers:
- In general, a length of 18-30 nucleotides for primers is good.
- Try to make the melting temperature (Tm) of the primers between 65 degrees C and 75 degrees C, and within 5 degrees C of each other.
- If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
- Typically, 3 to 4 nucleotides are added 5' of the restriction enzyme site in the primer to allow for efficient cutting.
- Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in TOPO cloning, the primers should not have a phosphate modification.
Read more about primer design tips and tools at https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html.
You may choose to do a two-temperature protocol when the annealing temperature is relatively high. In this case, you would combine the annealing and the elongation steps, i.e., both can occur together at a temperature >62 degrees C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.
You can try adding 5-10% DMSO, up to 10% glycerol, or 1-2% formamide or a combination of these to facilitate difficult templates. Note: the use of cosolvents will lower the optimal annealing temperatures of your primers.
A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.
Hot start is a way to prevent DNA amplification from occurring before you want it to. One way to do this is to set up the PCR reaction on ice, which prevents the DNA polymerase from being active. An easier method is a use a ‘hot-start' enzyme, in which the DNA polymerase is provided in an inactive state until it undergoes a high-heat step.
The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94-95 degrees C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50-65 degrees C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72 degrees C, allowing for the polymerase to bind and synthesize a new strand of DNA.
Both AmpliTaq Gold and Platinum Taq are hot-start enzymes that allow you to set up your reactions on the benchtop without the need for ice. AmpliTaq Gold is a chemically-modified hot-start enzyme, provided in an inactive state. Heat activates the enzyme, with full activity after 10 min at 95 degrees C. Platinum Taq is an antibody-mediated hot-start enzyme. The anti-Taq antibodies bind and inactivate the enzyme, until the heat denaturation step of the PCR reaction (30 sec to 2 min), which activates the enzyme.
With Platinum technology, anti-DNA polymerase antibodies bind to the enzyme until the denaturing step at 94 degrees C, when the antibodies degrade. The polymerase is now active and primer extension can occur. AccuPrime Taq combines Platinum Taq (Taq + Platinum antibodies) with proprietary thermostable AccuPrime accessory proteins. The 10X reaction buffer contains the accessory proteins which enhance specific primer-template hybridization during each cycle of PCR.
Please see the comparison below on the following criteria:
Enzyme, Relative Fidelity, Amplicon Length, and 3' Overhang (+/-)
Taq, 1, <5 kb, +
Platinum II Taq Hot-Start, 1, <5 kb, +
Platinum Taq, 1, <5 kb, +
AccuPrime Taq, 2, <5 kb, +
Platinum Taq HiFi, 6, <20 kb, +/-
AccuPrime Taq HiFi, 9 <20 kb, +/-
Platinum Pfx, 26, <12 kb, -
AccuPrime Pfx, 26, <12 kb, -
Pfx50, 50, <4 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
Taq DNA Polymerase has some reverse transcriptase (RT) activity at 68-78 degrees C. The processivity as a reverse transcriptase is 100-400 bp per min. The error rate can be up to 2%. (References: Jones M (1989) NAR 17:8387 and Shaffer A (1990) Anal Biochem 190:292).
Additionally, Mn++ can stimulate the RT activity of the Taq polymerase. (Reference: Myers and Gelfand (1991) Biochem 30:7661. This reference compares the RT activity of Tth to Taq.)
No, Platinum Taq DNA Polymerase is not a proofreading enzyme--it DOES NOT have 3' to 5' exonuclease activity. Platinum Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that inhibits polymerase activity in a temperature-dependent manner. Once the antibody is dissociated, the Platinum Taq DNA polymerase regains its full activity.
Platinum DNA Polymerase High Fidelity and Platinum Pfx polymerase are better choices when proofreading activity is desired.
Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.
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).
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.
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.
We have the following recommendations:
- Increase the extension time from 1 min/kb to 1.5 min/kb.
- Vary total PCR cycles from 20-40; 35 cycles is typical.
- Try denaturing at 95 degrees C for 45 seconds.
- Increase the amount of template for targets >5 kb.
- Use 2.5 U of Platinum Taq for each 50 µL reaction.
- GC-rich or problematic targets work better with MgSO4 instead of MgCl2.
- For GC-rich templates, test higher annealing temperatures starting with the temperature that is equal to your primer Tm and in increments of 2 degrees C for up to six different temperatures.
- Vary the KB extender solution from 1.5 µL to 4.5 µL per 50 µL reaction.
The KB Extender enhances the amplification of GC-rich and problematic sequences, and the extension of genomic DNA targets of >5 kb. The KB Extender lowers DNA melting temperature (Tm), and the co-solvent and amplification buffer offer higher primer specificity, broader Mg concentration and annealing temperature optima, as well as improved Taq thermostabilization. Please note that primer sets that generate specific products using standard PCR buffer may not benefit from using KB Extender. Excessive use of the KB Extender may reduce yield, particularly for non-GC rich amplicons. We generally recommend varying the KB Extender solution from 1.5-4.5 µL per 50 µL reaction.
Yes, we offer several Platinum Taq DNA polymerases and their master mixes, supplemented with tracking dyes for direct loading of PCR products on gels:
- Platinum II Hot-Start Green PCR Master Mix (2X)
- Platinum SuperFi Green DNA Polymerase and Platinum SuperFi Green PCR Master Mix
- Platinum Taq Green Hot-Start DNA Polymerase and Platinum Green Hot-Start PCR Master Mix (2X)
- DreamTaq Green Hot-Start DNA Polymerase and DreamTaq Hot-Start Green PCR Master Mix
- Phusion Green Hot-Start II High-Fidelity DNA Polymerase and Phusion Green Hot-Start II High-Fidelity PCR Master Mix.
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%.
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.
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.
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.
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.
Successful amplification of long PCR targets is dependent on 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. A few examples of our long PCR enzymes include our Elonagase enzyme mix that can be used for amplicons up to 30kb (blend of Taq and proofreading enzyme) or our Phire Hot Start II enzyme mix that can be used for amplicons up to 20 kb (Taq polymerase). Read more here: https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/long-fragment-pcr.html