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Primer design and Blast program at NCBI


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#1 Joscha

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Posted 11 January 2010 - 02:19 AM

Hello,

has someone of you experience with the Primer Blast program at the NCBI homepage. I tried several designs and it gave me some primers which where supposed to be specific for my template but when I Blast them again I can find quite a lot of possible misspriming possibilities. Additionally, when checking the primers with Primer 3 the 3'-end is often unacceptable because of too many G/C pairs. Primer Blast is using the search engine from Primer3 that's why I don't really understand these results.

One additional question would be the use of the thermodynamic parameters and the salt correction formula in Primer3. You can choose from Breslauer; Schilkraut & Lifson as well as SantaLucia for both. When using the parameters from SantaLucia the calculated primer Tm is lower. Ussually 60C is said to be optimal with the parameters from Breslauer and Schilkraut & Lifson. These primers have a lower Tm when recalculated with parameters from Santa Lucia (up to 10C). Do I also have to lower my optimal Tm for the design then? If yes how much do I have to decrease it?

Thanks in advance.

Joscha

Edited by Joscha, 11 January 2010 - 03:06 AM.


#2 susanna

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Posted 10 February 2010 - 08:20 AM

if your not sure about the tm, you can always do touchdown pcr,
but i actually dont think that it makes much difference, if you amplify at for instance 57C or 55C


Hello,

has someone of you experience with the Primer Blast program at the NCBI homepage. I tried several designs and it gave me some primers which where supposed to be specific for my template but when I Blast them again I can find quite a lot of possible misspriming possibilities. Additionally, when checking the primers with Primer 3 the 3'-end is often unacceptable because of too many G/C pairs. Primer Blast is using the search engine from Primer3 that's why I don't really understand these results.

One additional question would be the use of the thermodynamic parameters and the salt correction formula in Primer3. You can choose from Breslauer; Schilkraut & Lifson as well as SantaLucia for both. When using the parameters from SantaLucia the calculated primer Tm is lower. Ussually 60C is said to be optimal with the parameters from Breslauer and Schilkraut & Lifson. These primers have a lower Tm when recalculated with parameters from Santa Lucia (up to 10C). Do I also have to lower my optimal Tm for the design then? If yes how much do I have to decrease it?

Thanks in advance.

Joscha






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