The CleanAmp™ primers were created to reduce primer dimer and mis-priming however the CleanAmp group for the primers is situated on the internucleotide phosphate linkages.
This makes them very similar to a phosphorothioate or methyl phosphonate oligo which allows for nuclease resistance. I would recommend the precision primers since there are two CleanAmp groups for added nuclease stability. The CleanAmp primers 3’ OH group is exposed (not protected). The protecting group for the primers is different than for the CleanAmp dNTPs which is is blocking the 3’ OH.
The CleanAmp™ dNTPs have a half life of 5 minutes at 95°C in PCR buffer. At 92°C the deprotection rate will be slightly slower however this temperature should allow deprotection of enough of the CleanAmp™ dNTPs to work in your PCR. If needed, you could extend the time of the initial denaturation. Please contact us if you have any additional questions.
Yes. Some optimization may be required in your system.
The CleanAmp™ One-step RT-PCR Master Mix provides a Hot Start RT step, which prevents amplification of unwanted side reactions and truncated products due to RNA secondary structure. At 47°C, a small amount of denatured RNA template will be present to generate just enough of the correct cDNA to go into the PCR reaction. A key step in developing the CleanAmp™ One-step RT-PCR Master Mix was ensuring amplification of targets with high secondary structure, such as ABCA7 and PBGD.
The RT enzyme has an optimal temperature range of 42°C – 48°C. Due to the CleanAmp™ deprotection kinetics, we recommend temperatures at the higher end of that range, optimally 47°C.
Yes. We have successfully amplified up to 4 targets in the same reaction. Higher plexing may be possible.
Yes. In addition to primers, template RNA and water, add real-time reagents according to manufacturer’s recommendations (SYBR® Green, SYTO® 9, TaqMan® probes, ROX™, etc.)
CleanAmp™ dUTP is a dUTP analog which contains the CleanAmp™ thermolabile protecting group for improved PCR specificity.
Optimal PCR results are achieved when CleanAmp™ dUTP is in a three-fold excess over dATP, dCTP and dGTP. Use CleanAmp™ dUTP with standard dNTPs for well-behaved targets and with CleanAmp™ dNTPs for problematic targets.
CleanAmp™ dUTP can be used with uracil-N-glycosylase (UNG) in PCR-based carryover decontamination schemes. Please refer to the enzyme manufacturer for the recommended conditions for the UNG step of the reaction.
CleanAmp™ 7-deaza-dGTP and CleanAmp™ 7-deaza-dGTP Mix function well on their own for most sequences with <80% GC-content. However, should you have a problematic target or one that is >80% GC-content, addition of any one of these additives may increase specificity. We have found 0.5M Betaine or 10% Glycerol give the best results.
Preliminary results show CleanAmp™ 7-deaza-dGTP Mix functions with a variety of enzymes. However, we recommend Invitrogen™ Taq DNA polymerase for best results. For questions regarding your specific enzyme, please contact us.
CleanAmp™ 7-deaza-dGTP Mix contains the CleanAmp™ modified nucleoside triphosphates of dA, dC, (dG:7-Deaza-dG) and dT at 10 mM. The dG mix is a 1:3 ratio of CleanAmp™ dGTP:CleanAmp™ 7-deaza-dGTP.
For targets with <70% GC-content, addition of theCleanAmp™ 7-deaza-dGTP with standard dNTPs should amplify the target well. For targets with >70% GC-content, we recommend theCleanAmp™ 7-deaza-dGTP Mix for a robust amplification.
No, CleanAmp™ dNTPs are reverted back to the corresponding standard dNTP prior to being incorporated into the amplicon. The 3’-hydroxyl modification blocks enzyme incorporation until it is removed during heat activation. After heat activation, the enzyme efficiently incorporates the corresponding standard dNTP.
We are currently testing the kinetics of the deprotection of CleanAmp™ dNTPs at lower temperatures such as 60°C. Both the temperature and the pH of the PCR reaction affect the deprotection rate of the CleanAmp™ dNTPs where increased temperature and acidic conditions accelerate deprotection.
Our studies of deprotection at 55°C in 1X PCR buffer (pH 8.3) indicate that 40% of the dNTPs are deprotected after 2 hrs. Although deprotection is slower than at the elevated temperatures of PCR (95°C), adjustments to your reaction conditions, such as higher dNTP concentrations will likely allow for success.
Our CleanAmp™ Primers contain a different modification, but provide Hot Start activation using the same principle. At lower temperatures the rate of deprotection is slower, with a lower concentration of activated primers. To counter act the slower rate, we recommend using a higher concentration of primers.
CleanAmp™ dNTPs can be used with a broad selection of DNA polymerases including Taq, Pfu, Pfu (exo-),Dynazyme™, Deep VentR™, Tth and Tfi. When using CleanAmp™ dNTPs with these polymerases, all were able to produce the desired amplicon.
When standard cycling protocols are employed, a 2-5 minute initial denaturation step at 95°C allows for robust amplification. Recently we have found that initial denaturation times as short as 30 seconds can be used without any effect on PCR efficiency.
For handling stock solution, thaw for 5-15 minutes at room temperature. Do not thaw by heating. The unused portion should be returned to the freezer as quickly as possible. We recommend aliquotting stock solutions into several tubes to prevent extended room temperature exposure over many uses.
For best performance, we recommend distributing your stock solution into smaller aliquots that are sufficient for one week of work. To avoid prolonged exposure of the CleanAmp™ dNTP Mix stock solution to room temperature, store stock nucleotide solutions in the freezer at -20°C. We recommend not subjecting the CleanAmp™ dNTPs to more than 20 freeze-thaw cycles.
It contains the modified nucleoside triphosphates of dA, dC, dG, and dT. We currently offer CleanAmp™ dNTPs in a mix but the CleanAmp™ dNTPs are also available individually upon request. We have found that sometimes the replacement of just one or two of the natural nucleotides with CleanAmp™ dNTPs is enough to have the desired effect.
CleanAmp™ dNTPs improve PCR specificity, make PCR reactions cleaner, eliminate or reduce off-target amplicon formation, and are compatible with both Hot Start and non-Hot Start DNA polymerases that employ different buffer compositions (pH 7.5-9, 25°C). CleanAmp™ dNTPs are water soluble, stable for at least 1 year when frozen at –20°C and are inexpensive compared to other Hot Start technologies.
CleanAmp™ dNTPs are most stable in the shipped mix, diluted in PCR buffer (8-9 pH). In these conditions, they are stable for at least one year at –20°C. Exposure to higher temperatures during shipment does not pose performance risks. Avoid repeated freeze/thaw cycles and exposure to room temperature for more that 24 total hours. Upon first use, it is recommended to aliquot samples into single use portions.
CleanAmp™ dNTPs are able to decrease the formation of primer dimer products while increasing the specificity of the desired product better than unmodified dNTPs. Use of CleanAmp™ dNTPs also reduces data fluctuation, giving a more consistent PCR result.
CleanAmp™ dNTPs contain thermolabile modification groups that allow for a dNTP-mediated Hot Start activation approach in PCR. The introduction of temperature sensitive protecting groups onto the 3’-hydroxyl of a dNTP blocks primer extension at the less stringent, lower temperatures of PCR reaction preparation. When the reaction is heated to the elevated temperatures of PCR, the protecting group is removed to form the corresponding standard dNTP, which is now a suitable DNA polymerase substrate.
The stock solution is the DMSO solution in which your primers are shipped. Working solutions are any dilutions that are made using the stock, generally into aqueous solution.
The initial denaturation time is 2-10 minutes at 94°C for standard thermal cycling protocols. If used in combination with a chemically modified DNA polymerase, such as AmpliTaq Gold DNA polymerase, an initial denaturation of 10-20 minutes may be required. For fast cycling, shorter denaturation times at 94°C can be employed.
For handling stock solution: Thaw for 5-15 minutes at room temperature. Do not thaw by heating. The unused portion should be returned to the freezer as quickly as possible. We recommend aliquotting stock solutions into several tubes to prevent extended room temperature exposure over many uses. For handling working solutions: Dilutions of the stock solution should be made as needed, using water or aqueous buffer (pH 7-9) as the diluents, and then stored on ice. Working solutions should be used immediately.
Store at or below -20°C to avoid loss of hot start activity.
You do not need to use a DNA polymerase with CleanAmp™ Primers. If you would like to include a DNA polymerase, some validated sources of Taq DNA polymerase are as follows: Taq DNA polymerase (native and recombinant) from Invitrogen; Taq DNA polymerase (recombinant) from New England Biolabs; and EconoTaq™ DNA polymerase from Lucigen. Other validated DNA polymerases with comparable performance to Taq are: Pfu DNA polymerase (exo + and exo -) from Strategene; Dynazyme™ DNA polymerase from Finnzymes; Tth DNA polymerase from USB; Tfi DNA polymerase from Invitrogen; and Deep Vent™ DNA polymerase from New England Biolabs.
DMSO improves PCR efficiency by disruption of GC-rich sequences. Typically, 0-10% DMSO is introduced into the reaction. DMSO can also inhibit PCR performance, so be sure that the percent DMSO introduced by CleanAmp™ Primers is not inhibitory to the reaction. At high DMSO percentages, the performance of CleanAmp™ Primers may be reduced. Therefore we recommend the addition of DMSO into your reaction only if absolutely necessary and to a maximum of 2% volume per volume.
CleanAmp™ Primers can be employed over a much wider concentration range than unmodified primers. It is recommended that you perform a titration of your primers to determine the optimal concentration for improved Ct, with reduced off-target amplicon formation. Generally, primer template systems prone to mis-priming require a much lower primer concentration than those prone to primer dimer formation.
There are two forms, which differ in the rate of release of the protecting group. CleanAmp™ Turbo Primers are for fast cycling (<45 minutes) and multiplexed PCR. You will see improved amplicon yield and reduced mis-priming and primer dimer formation. CleanAmp™ Precision Primers are for standard cycling (>45 minutes). You will see improved specificity of amplification, improved limit of detection and the greatest reduction in mis-priming and primer dimer formation.
When in stock solution, they can be stored at room temperature for 48 hours, at 4°C for 60 days and at -20°C for 1 year. When in working solution, they can be stored at room temperature for 3 hours, at 4°C for 3 days and at -20°C for 20 days.
You will receive the typical synthesis yield of 10-15 ODs.
A. CleanAmp™ dNTPs can be used with a broad selection of DNA polymerases including Taq, Pfu, Pfu (exo-), Dynazyme™, Deep VentR™, Tth and Tfi. When using CleanAmp™ dNTPs with these polymerases, all were able to produce the desired amplicon. Learn More…
A. No, CleanAmp™ dNTPs are reverted back to the corresponding standard dNTP prior to being incorporated into the amplicon. The 3’-hydroxyl modification blocks enzyme incorporation until it is removed during heat activation. After heat activation, the enzyme efficiently incorporates the corresponding standard dNTP.
A. CleanAmp™ dNTPs are able to decrease the formation of primer dimer products while increasing the specificity of the desired product better than unmodified dNTPs. Use of CleanAmp™ dNTPs also reduces data fluctuation, giving a more consistent PCR result.
What is the expiration date of my modified nucleotide?
All of our modified nucleotides are submitted to quality control annually to ensure that they still meet our specifications, therefore we do not assign expiration dates to our modified nucleotides other than our CleanAmpTM dNTPs. Most modified nucleotides are stable for several years when stored properly at -20oC. Read More…
Have a question about modified nucleotides? Post a comment below.
What are the key benefits to new, improved CleanAmp™ dNTPs?
Improved Amplicon Yield in Endpoint PCR
– Earlier Cq Values in Fast Cycling Real-Time PCR
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Have a question about CleanAmp™? Post a comment below.
Will CleanAmp™ dNTPs activate at 60°C?
We are currently testing the kinetics of the deprotection of CleanAmp™ dNTPs at lower temperatures such as 60°C. Both the temperature and the pH of the PCR reaction affect the deprotection rate of the CleanAmp™ dNTPs where increased temperature and acidic conditions accelerate deprotection. Read More…
Have a question about CleanAmp™? Post a comment below.
I just wondered if it is possible to buy NTP’s (in particular ATP) that works like the CleanAmp™ nucleotides? A preferable version would be activated by light. – Leif Larsen, Vipergen
The CleanAmp™ modification can be introduced onto a number of NTP analogs, including ATP. This would allow you to control activation via pH or heat. There are other approaches to introduce light activatable modifications onto ATP as well. Read More…
Have a question about NTPs? Post a comment below.
Do you have any CleanAmp products that were determined to be product “failures” during your development? I know you made products with a hot start at high temperature a goal but do you have any chemistries that activated at ~56oC and were never released as viable product? Customized chemistries are of interest for ongoing development in my organization with an activation temp ~56oC. Thanks, – Dan Shaffer, Envirologix
Thank you for your interest in our CleanAmp™ Products. We did, as you thought, try several protecting groups before landing on the current CleanAmp™ chemistry … Read More
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We are proud to introduce the latest addition to the CleanAmp™ Product Line, CleanAmp™ 7-deaza-dGTP for GC-rich target amplification. When DNA targets high in GC content are amplified, PCR product formation can often be compromised by inadequate strand separation and the propensity for complex secondary structure formation. The inability of the DNA polymerase to negotiate through regions of strong secondary structure, such as hairpins, often results in the formation of truncated PCR products. In addition, mis-priming amplification products are prevalent when primers are high in GC content. Despite the inherent challenges, detection of GC-rich sequences is becoming increasingly important for the molecular diagnosis of inherited diseases. Read More…
I was researching your CleanAmp primers and would like to know if they could be activated later in the PCR cycles. For example, I have 2 pairs of primers for PCR and would like the first pair to work for cycle 1 through 10 followed by a long activation period to have both pairs working. What’s the probability that the 2nd pair would be activated during the first 10 denaturing cycles. – Chun-Nan Chen
Thank you for your question about CleanAmp Primers. For your interest in having one primer pair work earlier in PCR, with the second primer pair being activated after the first 10 thermal cycles, the best way to achieve this is to prepare the first primer pair without modifications and to prepare the second primer pair with CleanAmp Precision modifications… Read More
Have a question about CleanAmp™ Primers? Post a comment below.