Are the primers mainly reducing primer-dimer or they are also protected from exonuclease activity of proof-reading polymerase? In other words, is the 3’OH of the primer exposed or available to low temperature before they are heat inactivated.

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.

How should I handle CleanAmp™ Primers?

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.

Which DNA Polymerase should I use with CleanAmp™ Primers?

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.

Can I use DMSO with CleanAmp™ Primers in my reaction?

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.

What concentration should I use for CleanAmp™ Primers?

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.

How do I select the CleanAmp™ Primer type that I need?

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.