Do I Need to Work on Ice for Hot Start PCR?

In summary, it is generally recommended to work with everything on ice for PCR unless the protocol specifies otherwise. However, for Hot Start PCR, it is not necessary to work on ice as unspecific binding is eliminated during the first denaturation step. This eliminates the need for working on ice to reduce the activity of the polymerase and unspecific annealing of primers.
  • #1
sotellme
75
0
Do i need to work on ice when i do Hot start PCR?


Hope for inputs.

Thanks.
 
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  • #2
As a general rule, I work with everything on ice for PCR unless the protocol specifies otherwise.
 
  • #3
Yes, I always keep everything on ice too (best to be on the safe side :wink:)
 
  • #4
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.
 

Related to Do I Need to Work on Ice for Hot Start PCR?

1. What is Hot Start PCR and why is it important?

Hot Start PCR is a technique used in polymerase chain reaction (PCR) to prevent non-specific amplification of DNA. It involves temporarily inhibiting the activity of the DNA polymerase enzyme until the reaction is heated to the optimal temperature, thereby increasing the specificity and efficiency of the reaction. This is important because it helps to reduce background noise and improve the accuracy of the results.

2. Do I need to work on ice for Hot Start PCR?

Yes, it is recommended to work on ice during the preparation and handling of Hot Start PCR reactions. This is because the DNA polymerase enzyme used in Hot Start PCR is sensitive to heat and can become active at room temperature, leading to non-specific amplification. Working on ice helps to keep the reaction and enzyme cool, thereby reducing the chances of non-specific amplification.

3. Can I use a regular PCR machine for Hot Start PCR?

Yes, a regular PCR machine can be used for Hot Start PCR. However, it is important to make sure that the machine has the capability to maintain a low temperature (e.g. 4°C) during the initial steps of the reaction. This is necessary to prevent the DNA polymerase enzyme from becoming active prematurely.

4. What are the benefits of using Hot Start PCR?

The benefits of using Hot Start PCR include increased specificity and efficiency of the reaction, reduced background noise, and improved accuracy of the results. It also allows for the use of less template DNA and minimizes the chances of contamination, making it a valuable technique in molecular biology and diagnostic testing.

5. Are there any disadvantages to using Hot Start PCR?

One potential disadvantage of Hot Start PCR is the increased cost, as specialized enzymes or modified DNA polymerases may be required. Additionally, the extra steps involved in the technique may also increase the overall time and complexity of the reaction. However, the benefits of improved specificity and accuracy often outweigh these potential drawbacks.

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