Re: [DIYbio] How important is it for primers to have 40-60% GC content?

[Jeswin]

On Friday, May 03, 2013 11:02:06 PM, Zeb Haradon wrote:
>
> I did find some primers that have a "buffer" of only 167 base pairs,
> so I can run it next to a 100 bp ladder and it will be fairly easy to
> visualize (max size - a sample with 10 repeats - will be 647 bp). The
> problem is that the reverse primer has a GC content of 66.67%. What
> I'm reading online says to try to aim for 40-60%. So my question, for
> someone with more PCR experience than me (none) - how likely is it
> that this primer will work? Is there anything I can do to compensate
> for it (raise the temperature)? I can provide more detail if needed.
>

I haven't found it to be a big deal. I think most primers I used had at
least 50% G/C. I think the melting temperature is more important when
designing a primer.

[Nathan McCorkle]

Simply, the length and GC ratio influence the melting temp (Tm), less
simply the secondary, tertiary, maybe quaternary structure also
influence the Tm.

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--
-Nathan

[SC]

>I'm finding is that the buffer is much larger than I imagined

Can you explain what you mean by that?

As previously discussed, the primer you suggest should be OK, but you may need to adjust the TM a bit.