Primer3 design tool

  • Thread starter sobored
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When I use Primer3 design tool and the default Primer3 uses [Mg++] = 1.5 mM, whereas the average concentration is 5 mM in PCR kits. This concentration has a huge effect on the Tm. There is a “Salt concentration” box. Testing primer set designs for accuracy has shown that making the “Salt concentration” value 60 mM can help correct the Tm's.

My question is; when I set the “Salt concentration” at 60 mM should I also have to use salt in my reaction and what kind of salt ? and can I use other values than 60 mM salt?

Anyone who has used this tool before?
 

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  • #2
Monique
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You should use 1.5 mM MgCl2 in your PCR, that is the standard condition. The 5 mM in the PCR kits is a stock solution: you dilute the MgCl2 in your PCR mixture.

60 mM MgCl2 in your PCR? :bugeye: I don't think that's going to work!

Redesign your primers so that they have an acceptable Tm: their length should be around 20 bp and the GC content about 50%. Make sure to avoid primer dimers.
 
  • #3
Monique
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Btw, you can go up to 5 mM with your MgCl2 concentration. What will happen though is that the basepairing interaction of the DNA will be very strong, causing aspecific binding of the primers and thus the amplification of aspecific products.

If you are interested in whether MgCl2 is a rate-limiting step in your PCR, you should do a MgCl2 titration of say: 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM.

Also make sure that there is no EDTA in your buffer, it chelates Mg2+.
 

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