Predict second structure in PCR amplicon

2 replies [Last post]
ZZ
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Joined: 05/16/2011

Hello:

I am new to Mfold and try to use it to predict the second structures in PCR amplicon to help me design primers (real-time PCR)

I set up input as: fold at 60 °C. [Na+] = 50 mM, [Mg++] = 2.5 mM, oligo correction , because I design the primers annealing temperature 60°C. My first question: is this setup correct?

My second question is: how much the dG is acceptable in real-time PCR? for example, I got output "Structure 1 : dG = -2.58 kcal/mol" , Does this structure affect PCR efficiency?

Thank you in advance for your answer.
Sincerely,
ZZ

don123
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Joined: 02/23/2015
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Morismemo
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Joined: 07/27/2013
PCR

Have the same question as yours "how much the dG is acceptable in real-time PCR?" and after search found your post but not the answer!
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