1 reply to this topic

[Joscha]

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 60°C 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 10°C). 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.

[susanna]

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 57°C or 55°C

Joscha, on Jan 11 2010, 03:19 AM, said:

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 60°C 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 10°C). 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