End-Point PCR and PCR Primers Support - Getting Started

Recombinant Taq and native Taq polymerase are identical in terms of their activity, specificity, thermostability, and performance in PCR. Recombinant Taq has been expressed in a bacterial system and purified, whereas native Taq has been purified from the host.

We offer Platinum™ SuperFi™ DNA polymerase that displays greater than 100 times higher fidelity than Taq DNA polymerase.

Please see the following comparison table:

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

Yes, you can use a proofreading polymerase for PCR. However, you will need to add 3′ A-overhangs to your PCR product prior to TA cloning.

We would recommend using our Platinum II Taq Hot-Start DNA Polymerase, which is supplied with Platinum GC Enhancer, a special additive optimized to improve amplification of GC-rich targets (recommended for use with targets containing >65% GC). If it is important to preserve DNA sequence accuracy, we offer the high-fidelity Platinum SuperFi DNA Polymerase together with the SuperFi GC Enhancer to enhance the specificity and yield of difficult GC-rich target amplicons. AmpliTaq Gold 360 DNA Polymerase with the 360 GC Enhancer buffer has been shown to work with templates containing up to 80% GC. Alternatively, our PCRx Enhancer System can be used in conjunction with DNA polymerases, including native/recombinant Taq, Platinum Taq, and Platinum Taq High Fidelity to optimize PCR of problematic and/or GC-rich templates. 

We strongly suggest using MgSO₄. While MgCl₂ may work in some cases, MgSO₄ usually produces more robust and reproducible products, as sulfate is the best anion found for the Platinum® Taq High Fidelity enzyme.

With Platinum® technology, anti-DNA polymerase antibodies bind to the enzyme until the denaturing step at 94°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.

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.

Yes, the Platinum® Multiplex PCR master mix contains Platinum® Taq DNA Polymerase that leaves A overhangs on PCR products.

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°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.

Yes, the enzyme mix leaves 3′ A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3′ A-overhangs to the product for TA cloning.

In our experience, the Gold Buffer gives the best performance. Here are the formulations:
GeneAmp® 10X PCR Buffer: 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl₂, pH 8.3, 0.01% (w/v) gelatin
GeneAmp® 10X PCR Buffer II*: 100 mM Tris-HCl, 500 mM KCl, pH 8.3
GeneAmp® 10X PCR Buffer Gold*: 150 mM Tris-HCl, 500 mM KCl, pH 8.0
*Buffer comes with separate MgCl₂ Solution (25 mM).

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. 

Yes the green dye is compatible with downstream applications such as fluorescent automatic DNA sequencing, ligation, and restriction digestion.

We recommend using the GC enhancer for targets with >65% GC sequences. We recommend that you have a final concentration of 20% of the GC enhancer in the reaction mix. There is enough GC enhancer included in the kit to add it to every PCR 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.

The final concentration is 1.5 mM MgCl2.

Platinum SuperFi DNA Polymerase significantly differs from many other DNA polymerases, therefore annealing rules should be adjusted by using our Tm calculator. Due to high processivity, Platinum SuperFi DNA Polymerase also has shorter cycling times than many other DNA polymerases.

No, primers with various melting temperatures can be used. If designed following the general primer design rules, the majority of primers will anneal specifically at 60 degrees C regardless of their melting temperature.

5X Platinum II PCR Buffer contains isostabilizing molecules that increase primer-template duplex stability during the annealing step and contribute to enhanced specificity. As a result, we expect most primer pairs to anneal at 60 degrees C in this polymerase/buffer system, eliminating the need for annealing temperature optimization.

Usually, primers that are well designed and work in PCR with standard Taq under standard PCR conditions, anneal specifically in Platinum II PCR buffer at 60 degrees C regardless of their Tm. If amplification of a particular template-primer pair does not give satisfactory results, we recommend redesigning the primers.

If there is no possibility for redesign, use a temperature gradient and empirically determine the optimal annealing temperature. Start with an annealing temperature that is at least 5 degrees C lower than your primer Tm.

Platinum II Taq Hot-Start DNA Polymerase contains engineered Taq DNA polymerase with increased DNA synthesis rate of 15 sec/kb at 68 degrees C extension temperature. Conveniently, the extension step can be prolonged up to 1 min/kb without a negative effect on specificity. This allows the cycling of shorter and longer amplicons together using the same protocol.

Yes, simple amplicons up to 1 kb with 45-65% GC sequences can be synthesized using a 2-step cycling protocol with a combined annealing/extension step at 60 degrees C. With the 2-step protocol, a denaturation step is performed for 5 sec at the increased temperature of 98 degrees C. For longer, GC-rich, and complex amplicons, or cDNA targets, we recommend using a 3-step cycling protocol.

Platinum Taq DNA Polymerase, DNA-free is manufactured using closed and single-use system technology to minimize DNA contamination risk. Platinum Taq DNA Polymerase, DNA-free provides the same high level of performance and lot-to-lot consistency that is expected for the standard Platinum Taq DNA Polymerase.

Platinum Taq DNA Polymerase, DNA-free is certified to contain low-DNA contamination level: ≤0.01 copy of bacterial gDNA per enzyme unit; ≤0.001 copy/enzyme unit of human gDNA; ≤0.01 copy/enzyme unit of plasmid DNA.

Amplification signal in “no-template controls” (NTC), also known as false-positive signal, or background amplification in “reagent-only control”, may arise from contaminating DNA that entered control reactions from the environment, from the researcher, or was introduced into the PCR via contaminated PCR reagents or consumables. To avoid contaminating DNA in your experiments, it is important to follow these recommendations:

- Be cautious to maintain a DNA-free environment during handling and opening the vials with DNA polymerase, PCR buffer, and MgCl2
- Ensure that all components of the PCR, like dNTPs, primers, probes, or water are free of contaminating DNA that can give false positive PCR results
- Avoid opening the tubes containing DNA-free reagents multiple times to minimize risk of DNA contamination
- Work in a UV-irradiated workstation
- Use certified DNA-free pipette tips and PCR consumables

Platinum SuperFi DNA Polymerase accurately amplifies long fragments (up to 20 kb) with high yields and specificity. Amplification of even larger fragment sizes up to 40 kb has been demonstrated, but may require additional optimization of reaction conditions and primer design.

No. Platinum SuperFi DNA Polymerase generates blunt ended products.

No. Platinum SuperFi DNA Polymerase cannot read uracil in the template strand, therefore, it is not recommended for use with bisulfite-converted DNA.

No. The colored buffer does not interfere with PCR performance and does not change enzyme features.

All Platinum SuperFi product formats are supplied with 5X SuperFi GC Enhancer which is optimized to improve amplification of GC-rich targets (recommended for use with targets containing >65% GC content)

Yes. To improve amplification of AT-rich targets, we recommend reducing the extension temperature to 68 degrees C or add 5-15 mM Tetramethylammonium Chloride (TMAC).

Good lab practices are important for long fragment amplification. These include using high-quality templates (pure, fresh and intact) and fresh primer solutions. Optimization steps to consider include decreasing denaturation and extension temperatures, lengthening extension times as recommended in the manual, and increasing template amounts.

Platinum SuperFi DNA Polymerase cannot read dUTP-derivatives or dITP in DNA templates, so the use of these analogues is not recommended.Platinum SuperFi DNA Polymerase can incorporate 7-deaza-dGTP and radiolabeled dNTPs.

Yes. DreamTaq Hot Start DNA Polymerase generates 3'-A overhangs.

DreamTaq Hot Start DNA Polymerase amplifies up to 6 kb from human genomic DNA and up to 20 kb from lambda DNA with high yields and specificity. Amplification of even longer fragments up to 9 kb from human genomic DNA has been demonstrated, but may require additional optimization of reaction conditions and primer design.

Yes. DreamTaq Hot Start DNA Polymerase can read uracil in the template strand and therefore can be used for amplification of bisulfite converted DNA.

Yes, DreamTaq Buffer (Cat. No. B65, 4 x 1.25 mL) and DreamTaq Green Buffer (Cat. No. B71, 4 x 1.25 mL) are available as stand-alone products.

Yes. 10X DreamTaq Buffer and 10X DreamTaq Green Buffer are the same for both polymerases.

DreamTaq Hot Start DNA Polymerase can incorporate dUTP and a variety of modified nucleotides, such as: dITP, 7-deaza-dGTP, fluorescein-12-dUTP, Biotin-11-dUTP, dm5CTP, alpha-thio-dCTP, and aminoallyl-dUTP.

Both linear and circular DNA templates can be amplified by Phi29 DNA Polymerase.

Both linear and circular DNA templates can be amplified by EquiPhi29 DNA Polymerase.

The amplification efficiency of MDA (multiple displacement amplification) reaction rapidly diminishes as the molecular weight of the starting material decreases, thus making the polymerase unsuitable for amplification of low-molecular weight DNA. However, it is suitable for amplification from extremely low amounts of starting DNA material, making it ideal for single cell analysis.

EquiPhi29 DNA Polymerase is a thermostable enzyme. The optimal reaction temperature range is 42-45 degrees C, however it will also amplify DNA at 30 degrees C.

Yes, the reaction volume can be scaled down to 10 µL and scaled up to 50 µL from the recommended 20 µL. Scaling up the reaction volume more than 4-fold is not generally recommended.

Yes, we recommend inactivating the polymerase at 65 degrees C for 10 min.

No. dNTPs can be purchased separately (Cat. No. R0181).

No. dNTPs can be purchased separately (Cat. No. R0181).

Yes, 100 mM DTT is supplied as a separate component. The enzyme requires active reducing reagent in the reaction mix, therefore fresh DTT should be added separately. As DTT degrades over time, older DTT or DTT that has been frozen and thawed more than 10 times should be substituted with freshly prepared DTT.

Yes. The recommended protocol is supplied in the manual (https://www.thermofisher.com/order/catalog/product/A39390). Alternatively, amplification from single cells using EquiPhi29 DNA polymerase can be reviewed in the literature:

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.
Authors: Stepanauskas R, Fergusson EA, Brown J, Poulton NJ, Tupper B, Labonté JM, Becraft ED, Brown JM, Pachiadaki MG, Povilaitis T, Thompson BP, Mascena CJ, Bellows WK, Lubys A
Journal: Nat Commun 2017; 8(1):84
PubMed ID: 28729688

Phusion Green and Phire Green buffers contain two dyes for monitoring electrophoresis progress. The blue dye migrates with 3-5 kb DNA fragments and the yellow dye migrates faster than 10 bp DNA fragments in 1% agarose gel. The dyes have excitation peaks at 424 nm and 615 nm, respectively.

Yes. The dyes do not interfere with downstream applications such as DNA sequencing, ligation, and restriction digestion.

FFPE tissue, FFPE tissue sections, FFPE tissue blocks and microscope slides can be used with Platinum Direct PCR Universal Master Mix. DNA is highly fragmented in FFPE samples, and DNA quality being one of the main factors for successful amplification, we recommend amplifying fragments up to 300 bp with this master mix.

In order to obtain best results when amplifying DNA directly from a sample using Platinum Direct PCR Universal Master Mix, it is important to use a very small amount of starting material. The recommended size is 1-2 mm in diameter. Possible tools for sample handling are: tissue puncher, scalpel or 100 µL pipette tip for soft samples.

No, a tissue puncher is not provided with the kit. If a tissue puncher is not available, cut the sample with a scalpel. Tweezers, scalpel and other tools need to be cleaned with 2% NaClO solution to avoid cross contamination.

If your sample is larger than 1 mm in diameter, we recommend increasing the Lysis Buffer volume to make sure that the sample is completely covered in the buffer. Add Proteinase K accordingly.

For the Direct protocol, the recommended sample size is up to 1 mm. For the Lysis protocol, the recommended sample size is up to 2 mm. Sample size can be increased up to 1 cm, however, Lysis Buffer volume needs to be increased to cover the whole sample.

Samples can be incubated for up to 8 hr at room temperature in the Lysis Solution, prior to the 98 degrees C Proteinase K inactivation step. Please note that longer incubation may increase the product yield. For longer incubation, the Proteinase K inactivation step may be increased up to 10 min.

Electrophoretic separation on E-Gel agarose gels depends on the salt concentration in the analyzed sample. For optimal band separation, we recommend diluting PCR reactions 2- to 20-fold with water, prior to running on E-Gel agarose gels. The dyes in the Platinum Direct PCR Universal Master Mix do not interfere with fragment separation on E-Gel agarose gels.

Proteinase K is required when PCR is performed directly from tissue samples using the Direct PCR protocol. Cell debris present in these PCR products can cause DNA fragments to get trapped in the agarose gel wells and Proteinase K helps to eliminate this.

You can use any plant or tissue sample. However, when working with new sample materials, we recommend using the Lysis protocol as it allows several PCR reactions to be performed from the same sample during optimization. We also recommend having a positive control with purified sample DNA to ensure that the PCR conditions are optimal. If the positive control with purified DNA fails, the PCR conditions should be optimized before continuing further.

Platinum SuperFi II DNA Polymerase can amplify targets with high GC content (up to 75% GC) without any additional DNA melting agents. In cases of extremely GC-rich targets (>75% GC), addition of DMSO to final concentration of 5% is recommended.

Platinum SuperFi II DNA Polymerase enables amplification of both AT-rich and GC-rich targets. Lower extension temperature (68 degrees C or even 65 degrees C) can be helpful for extremely AT-rich targets.

Platinum SuperFi II DNA Polymerase cannot read dUTP-derivatives or dITP in DNA templates, so the use of these analogues is not recommended. Platinum SuperFi II DNA Polymerase can incorporate 7-deaza-dGTP and radiolabeled dNTPs.

The green tracking dye (which consists of a blue and a yellow dye) does not interfere with electrophoresis in E-Gel agarose gels. The sample should be diluted 2- to 20-fold for optimal separation using E-Gel agarose gels.

The green tracking dye (which consists of a blue and a yellow dye) is compatible with downstream applications such as fluorescent automated DNA sequencing, ligation, and restriction digestion. For applications that require analysis of PCR products by absorbance or fluorescence excitation, we recommend using colorless versions of the products (Platinum SuperFi II DNA Polymerase or Platinum SuperFi II PCR Master Mix) or purifying the PCR products prior to analysis.

Platinum SuperFi II reaction buffer is compatible with restriction digestion directly after PCR. Note that Platinum SuperFi II DNA Polymerase is not inactivated during PCR, thus purification of PCR products before restriction digestion is recommended if intact 5'- or 3'- overhangs are needed for cloning.

Platinum SuperFi II DNA Polymerase produces blunt-ended PCR products that can be cloned directly into blunt-ended cloning vectors. TA cloning is also possible if 3' dA-overhangs are added after PCR. We recommend purifying the PCR products of Platinum SuperFi DNA Polymerase before adding the overhangs. The procedure for adding 3' dA-overhangs (TA cloning) includes the following steps:
- Purify the PCR product (e.g., with a PCR purification kit or phenol extraction/DNA precipitation). Before adding the overhangs, PCR product must be purified, as the strong proofreading activity of any remaining Platinum SuperFi DNA Polymerase will degrade the added 3' dA-overhangs.
- Perform 3' dA addition with a Taq DNA polymerase.
Reaction components:
Purified PCR product
0.2 mM dATP
1x Taq Buffer
1 U Taq DNA Polymerase
Incubate the reaction for 20 min at 72 degrees C.
- Proceed to TA cloning. For optimal ligation efficiency, we recommend using fresh PCR products, since 3' dA-overhangs can be lost during storage

<p>Yes, Platinum SuperFi II DNA Polymerase works at both universal 60 degrees C annealing temperature and at annealing temperatures calculated with a Tm calculator. </p>

Good lab practices are important for long fragment amplification. These include using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Reducing primer concentration to 0.2 µM may also improve the results.

The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94–95°C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50–65°C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72°C, allowing for the polymerase to bind and synthesize a new strand of DNA.

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 MgCl₂ should be optimized for each template and primer pair. In general, the final concentration varies between 0.5–2.5 mM (when using 0.2 mM dNTPs). Note: EDTA or excess dNTPs can inhibit amplification by chelating the magnesium ions necessary for Taq DNA polymerase activity.

While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we’d recommend starting with 10% of the first-strand reaction for your PCR reaction.

Touchdown PCR involves decreasing the annealing temperature by 1°C every second cycle to a ‘touchdown’ annealing temperature which is then used for the remaining cycles. Touchdown PCR increases specificity and reduces background amplification. By starting at a high annealing temperature, only your gene of interest is amplified, allowing the target product to accumulate. Decreasing the annealing temperature through the remaining PCR cycles permits efficient amplification of the specific template.

Nested PCR requires two separate amplifications—the first one using one set of PCR primers and the second one using internal "nested" primers plus 1% or less of the first PCR reaction as a template. Nested PCR is used when the target is present in low abundance or when nonspecific PCR products are being produced along with the specific product. Semi-nested PCR is used when there is only enough sequence information to make a primer internal to one end of the primary PCR product such as in RACE (rapid amplification of cDNA ends).

Typically, primer annealing temperature is 3–5°C lower than the lowest primer melting temperature. If the temperature is too high, primers anneal poorly. If the temperature is too low, nonspecific annealing can occur.

A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.

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.

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°C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.

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°C and 75°C, and within 5°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 here.

Yes. OligoPerfect™ Designer can be used to design primers for sequencing, cloning, or detection.

Centrifuge the tube for a few seconds to collect the oligonucleotide at the bottom of the tube. Carefully open the tube, and dissolve the oligonucleotide in the appropriate volume of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). TE is recommended over deionized water since the pH of water is often slightly acidic and can cause hydrolysis of the oligonucleotide. It is also best to pipette the solution up and down at least 10 times. Please visit this webpage for more information on how to calculate primer concentration and resuspension volume.

Oligonucleotides from Thermo Fisher Scientific come lyophilized. It is recommended to store lyophilized (and reconstituted) oligos at -20°C. Lyophilized oligonucleotides are stable at -20°C for at least 1 year. Oligonucleotides dissolved in TE are stable for at least 6 months at -20°C or 4°C. Dissolved in water they are stable for at least 6 months at -20°C in the absence of nucleases. Lyophilized oligos should be stable for at least a few months at RT. Oligos in solution are stable for at least 6 months if they are stored at -20°C at a concentration greater than 10 μM. If the oligos are stored at 4°C, it is important that they are resuspended in TE, NOT water. (Note: at 4°C in water, the oligo will hydrolyze over time). AP and HRP conjugates are shipped in their respective storage buffers. Please store them at 4°C.

Depending on your specific application, a different level of purification may be required. Please refer to this table.

Oligos are made using a DNA synthesizer which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.

1. Deblocking: the first base, attached to the solid support via a chemical linker arm, is deprotected by removing the trityl protecting group. This produces a free 5′ OH group to react with the next base.
2. Coupling: the next base is added, which couples to the first base.
3. Capping: any of the first bases, which fail to react, are capped. These failed bases will play no further part in the synthesis cycle.
4. Oxidation: the bond between the first base and successfully coupled second base is oxidized to stabilize the growing chain.
5. Deblocking: the 5′ trityl group is removed from the base, which has been added.

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 for the minimum yield guarantees we offer for our oligos.

Coupling efficiency is the major factor affecting the length of DNA that can be synthesized. Base composition and synthesis scales will also be contributing factors. At 99% coupling efficiency, a crude solution of synthesized 95-mers would contain 38% full-length product and 62% (nx) failure sequences. This is before other chemical effects have been taken into account such as depurination. Depurination mainly affects the base A. The frequency of depurination is small but will increase significantly with primer length. For these reasons, we specify a maximum length of 100 bases, which we believe is the maximum length that can be synthesized routinely and economically.

Coupling efficiency is a way of measuring how efficiently the DNA synthesizer is adding new bases to the growing DNA chain. If every available base on the DNA chain reacted successfully with the new base, the coupling efficiency would be 100%. Few chemical reactions are 100% efficient. During DNA synthesis, the maximum coupling efficiency obtainable is normally around 98–99% (99% is typical). This means that at every coupling step, approximately 1–2% of the available bases in the chain fail to react with the new base being added. An approximation of the percentage of full-length oligonucleotide is obtained by the coupling efficiency raised to the power of its length (i.e., number of cycles), e.g., 0.99²² x 100 = 80% full-length primer. You can have your primers further purified to 95% full-length. Purification is highly recommended for long oligos. For example, a 64-mer synthesis will yield 0.99⁶⁴ x 100 = 53% full-length. This will only be obtained if the efficiency is 99% at every cycle.

The trityl group is colorless when attached to a DNA base but gives a characteristic orange color once removed. The intensity of this color can be measured by UV spectrophotometry and is directly related to the number of trityl molecules present. By comparing the absorbance of trityl releases throughout synthesis, it is possible to calculate the percentage of bases coupling successfully and hence the coupling efficiency.

Coupling efficiency is important as the effects are cumulative during DNA synthesis. The table 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.

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.

Please take a look at this list 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@lifetech.com to see if we can accommodate your request.

Unless you specifically request that primers be supplied in solution, they are shipped lyophilized. We recommend reconstituting them in the appropriate volume of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

We offer TE Buffer, pH 8.0 (Cat. Nos. AM9849 and AM9858), that consists of 10 mM Tris (adjusted to pH 8.0 with HCl) and 1 mM EDTA.

Yes, we can synthesize primers with degenerate bases. Click here for the electronic ordering code to use.

No, the primer will not have this modification. You must specify that it should have a 5’ phosphate modification.

A common equation used to calculate primer Tm is as follows:

Tm (in °C) = 2 (A+ T) + 4 (G + C)

An important parameter for primers is the melting temperature Tm. This 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°C to 70°C. Annealing temperatures are generally about 5°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.

A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.

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.

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°C. The advantage of a two-temperature protocol is that it is considerably quicker in comparison to the conventional three-temperature protocol.

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.

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.

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°C to 70°C. Annealing temperatures are generally about 5°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.

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.

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.

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.