How to obtain PCR product with NH2 group at the 5'end?

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No title

Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Paul Rutland

Changes at the 5' end of primers do not usually make much difference to pcr so I would treat this just like any normal pcr that fails to amplify. Check the presence of primer dimers, check the product CG composition and consider using additives like betaine or dmso to change secondary structure. Run a gradient of annealing temperatures and if all else fails try a different Taq enzyme

Aparna Sekhar

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Ajith Kumar

Hi,

Modification at 5' end should not cause problem in the PCR. Do you have the same primer without NH2? Please run both and see whether you are getting the product.