- #1
sotellme
- 75
- 0
Do i need to work on ice when i do Hot start PCR?
Hope for inputs.
Thanks.
Hope for inputs.
Thanks.
Click For Summary
Discussion Overview
The discussion centers on whether it is necessary to work on ice during Hot Start PCR (HS-PCR). Participants explore the implications of temperature management on the PCR process, particularly regarding enzyme activity and primer binding.
Discussion Character
- Debate/contested
- Technical explanation
Main Points Raised
- Some participants suggest that working on ice is a general practice for PCR to ensure optimal conditions, unless specified otherwise in the protocol.
- Others argue that it is not necessary to work on ice for HS-PCR, explaining that the initial denaturation step effectively eliminates unspecific primer binding, thus reducing the need for temperature control during setup.
- A participant emphasizes that the purpose of working on ice in standard PCR is to minimize polymerase activity and prevent unspecific annealing of primers, which may not be as critical in HS-PCR.
Areas of Agreement / Disagreement
Participants express differing views on the necessity of working on ice for HS-PCR, with no consensus reached on the best practice.
Contextual Notes
Some assumptions about the effectiveness of the initial denaturation step in HS-PCR and the role of temperature in enzyme activity and primer binding are discussed but remain unresolved.
- #2
Moonbear
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As a general rule, I work with everything on ice for PCR unless the protocol specifies otherwise.
- #3
Monique
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Yes, I always keep everything on ice too (best to be on the safe side 
)
)- #4
iansmith
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You do not have to work on ice for HS-PCR. The idea of working on ice when you are not doing HS-PCR is to reduce the activity of the polymerase and to reduce unspecific annealing of the primers. This is usually sufficient to elimination most of the unspecific binding problems.
For HS-PCR, the unspecific binding are eliminated because any unspecific annealed primers has been removed during first denaturation step. So when you add Taq after the denaturation step, unspecific annealed primers should not be present.
For HS-PCR, the unspecific binding are eliminated because any unspecific annealed primers has been removed during first denaturation step. So when you add Taq after the denaturation step, unspecific annealed primers should not be present.
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