- #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
SUMMARY
Working on ice is not necessary for Hot Start PCR (HS-PCR). The primary purpose of using ice in standard PCR is to minimize polymerase activity and prevent non-specific primer annealing. In HS-PCR, non-specific binding issues are addressed during the initial denaturation step, which effectively removes any non-specifically annealed primers before Taq polymerase is added. Therefore, maintaining samples on ice is not required for HS-PCR protocols.
PREREQUISITES- Understanding of Hot Start PCR (HS-PCR) mechanisms
- Knowledge of Taq polymerase function and behavior
- Familiarity with PCR denaturation steps
- Basic principles of primer annealing and specificity
- Research the differences between standard PCR and Hot Start PCR techniques
- Learn about the role of Taq polymerase in PCR reactions
- Explore methods to optimize primer design for HS-PCR
- Investigate common troubleshooting techniques for PCR amplification
Researchers, molecular biologists, and laboratory technicians involved in PCR optimization and protocol development will benefit from this discussion.
- #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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