PCR Hot start PCR - Mammalian DNA

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. However, non-specific product amplification and primer-dimer formation during set-up are major causes of PCR failure. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction

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4 years ago

4 years ago by Maryam Amir Qatar

When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?

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Discussion

4 years ago

4 years ago by Janina Reinhardt Switzerland

Hot start PCR using phusion green DNA polymerase

I am currently performing hot start PCR using phusion green hot start DNA polymerase in order to amplify a 8.8kb insert. However, I cannot get any amplified product at all. I have tried changing some parameters but I am not sure what to do. Any help?

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Found 5 matching solutions for this experiment

Q5® Hot Start High-Fidelity 2X Master Mix

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	Always thaw on ice

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
Always thaw on ice