SAGE questions

Bas Jansen bas at nospam.nl
Fri Nov 17 03:49:34 EST 2000


Hi Netters,

<odda at my-deja.com> schreef in bericht news:8v0vvm$g4r$1 at nnrp1.deja.com...
> Hi Olivier,
>
> The reason for taking two different linkers with two different primer
> binding sites is that during the ditag pcr step ditags with the same
> linker sequence can not be (efficiently) amplified. After the
> denaturation step, instead of annealing to the primers, they can also
> anneal to the second linker in the same single-stranded ditag molecule
> and form a loop, but will not be amplified by Taq Polymerase.
> Just check this by ligating Linkers to each other to form Linker 1 and
> Linker 2 homodimers and a Linker 1 - Linker 2 heterodimer and PCR
> amplify them. You will see, that only the heterodimer will be amplified.

Not necessarily. I have done that, and in my case there was amplification of
homodimers, albeit at lower levels. But suppression of amplification was far
from impressive. I have fiddled around with PCR conditions, and was
surprised that, when using AmpliTaq Gold in its commercial buffer, this
suppression was nearly complete as opposed to a PCR using AmpliTaq in the
exotic SAGE buffer.

[...]

Bas
basjhj at baserv.uci.kun.nl








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