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Asking about Hot Start PCR

Asking about Hot Start PCR - PCR - Polymerase Chain Reaction Forum

Asking about Hot Start PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 10-14-2009, 06:24 AM
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Default Asking about Hot Start PCR



Dear,

Can you explain about Hot Start PCR detail? Can I use normal DNA Taq polymerase in hot start pcr program?
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Old 10-14-2009, 04:31 PM
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Default Re: Asking about Hot Start PCR

"Hot-start" is basically a method to prevent non-specific amplification during the early stage of PCR, ie. as the temperature starts climbing up to 94-95 for the initial denaturation.

"Hot-start" DNA polymerases are enzymes that only become active after the initial denaturation. This usually involve the binding of antibodies/beads/etc to the enzyme that inhibits it, and will only dissociate from the enzyme at a hight temperature of 94-95. In other words, no amplification can occur as the PCR machine ramps up the temperature to 95 for the initial denaturation.

You can also run a "hot-start" PCR manually, eg:
1. Leave out a critical component of the PCR, eg Mg or Taq until the PCR machine reaches 94. Then only you add the final component.
2. Set a longer period for the initial denaturation. After the PCR machine ramps up to 94, then only you place your samples on the machine.
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Old 10-15-2009, 03:42 AM
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Default Re: Asking about Hot Start PCR

Hot-start has been explained by jonoave.

And yes, you can start hot-start PCR with a normal taq polymerase. What I normally do is to prepare my PCR samples as per normal, except that I miss out Taq. I leave my samples in the PCR machine and let the samples stay at 95degcel before I add in Taq to each sample and let the reaction continue.
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Old 10-15-2009, 11:34 AM
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Default Re: Asking about Hot Start PCR

hello dear

Hot Start PCR is same as normal PCR but the diffference lies in the addition of Taq pol here polymerase is added after denaturation has taken place. It is basically to prevent non-specific amplification. I hope this answers yours question please feel free to ask.
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