Re: [DIYbio] PCR Question

it's possible that the newly created strand from the first two primers
would then actually prime the KanR area as the forward primer... seems
like you could easily simulate the outcome of this

On Mon, Jan 7, 2013 at 12:53 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
> except the middle primer you envision is going toward the GFP gene,
> whereas it needs to go toward the KanR gene... remember 5' to 3' is
> the way the enzymes read/elongate DNA
>
>
> On Mon, Jan 7, 2013 at 12:49 PM, Mega <masterstorm123@gmail.com> wrote:
>> Hi everybody.
>>
>>
>> I'd just like to know if you could possibly do a PCR from two sources at a
>> time using three primers?
>>
>>
>>
>> So e.g.:
>>
>> Usually if you want to have a GFP-KanR construct, you would use four
>> primers, with the primers between the two genes of interest having the same
>> restriction site. Those would then be ligated later.
>>
>> What if you took just one primer which contains the end of GFP + a short
>> linker + beginning of KanR and of course one GPF starting primer and one
>> KanR end primer? Would that still work?
>>
>> Additionally, the sources were two different plasmids. Could that possibly
>> work, to get one construct in one step (without the need of restriction
>> digestion and re-ligating)
>>
>> May it be a problem that the ends of primers can have a false sequence?
>>
>>
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>
>
>
> --
> -Nathan



--
-Nathan

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