Primer

[Linden]

Hey guys,

I am designing a 40 bp primer (it is for homologous recombination in yeast), but part of it also anneal at another part of the plasmid, just like the drawing attached. Could it be a big issue? I dont have another plasmid with this gene..

Thanks a lot!

[Jeswin]

What's the size difference between the target fragment and the unwanted larger fragment? You could run one PCR and isolate your target fragment. Then use that DNA as template for a second PCR amplification.

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[Linden]

Thanks, Phillyj

Maybe I could digest it out of the plasmid, run an agarose gel, purify and then amplify. Probably ok, right?

[Jeswin]

On Mon, Jun 15, 2015 at 2:00 PM, Linden <ff.lin...@gmail.com> wrote:
>
> Thanks, Phillyj
>
> Maybe I could digest it out of the plasmid, run an agarose gel, purify and then amplify. Probably ok, right?
>

I don't know if you want to digest the plasmid and then PCR. It might
be easier to do PCR on the plasmid and then cut out the smaller
(Target) band. I am assuming that you get 2 bands with good
resolution. You may end up with a smear on the gel (after 1st PCR) so
then maybe a plasmid digest would help isolate the specific region you
want.

[Mega [Andreas Stuermer]]

The short band may be amplified prefferably by the reaction?
Do you have the sequence of the entire thing?

Digestion is a good idea, if there is a restriction site inbetween....