Re: [DIYbio] Re: *experienced* beginner is creating a bioluminescent plant - both synbio and old-fashioned way

Yea, 95 should be enough but for antibody-blocked pols you'll need an
"activation" initial denaturation of maybe 5 mins first. Not too long,
or you'll start deaminating the T's in your DNA.

What's the program vs. amplicon length and primer melting temperatures?
How much Magnesium are you adding, if it's not already in the buffer?

On 05/01/2013 03:22 PM, Mega wrote:
> The PCR didn't yield any fragments (but the ~50 bp Primers). Both from
> Genomic V.Fischeri (pure culture from Deutsche Sammlung
> Mikroorganismen) and pVIB.
>
>
> So we did some troubleshooting, and noticed that the error is very
> likely that my professor programed the thermcycler to 95°C instead of
> the 98°C mentioned many times in the description. At that temperature,
> the antibody from the Phusion polymerase still inhibits its work, we
> assume.
>
>
> At the time, he told me that it's just for denaturing, and 95°C would be
> absolutely sufficient. Seems not.
>
>
>
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