PCR Hot start PCR - Bacterial 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 hot-start DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction

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5 years ago

5 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

5 years ago

5 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 6 matching solutions for this experiment

TEMPase Hot Start Master Mix BLUE

Ampliqon

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	Working on ice is not required.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
Working on ice is not required.