Re: [DIYbio] PCR Question

you need a forward AND a reverse primer. If you encode the forward
primer for the KanR in the reverse primer of the GFP, then the forward
primer won't 'come alive' until the GFP gene has gone through PCR
twice. Once for the reverse GFP primer to extend, and once for the
forward GFP primer to extend ON THE NEW STRAND FORMED BY THE REVERSE
PRIMER. This resulting strand will be the correct sense to act as a
the forward primer for KanR.

I guess you'd only want a very small amount of the middle primer, to
avoid too many GFP-only amplifications.

I don't understand why your prof would use TATATATA at the end of a
primer unless they wanted to add that sequence to their amplicon, or
they hoped it would aide priming if the primer didn't match perfectly.

On Mon, Jan 7, 2013 at 1:07 PM, Andreas Sturm <masterstorm123@gmail.com> wrote:
> So you mean, the problem is that the GFP then is not necissarily amplified?
>
>
>
> On Mon, Jan 7, 2013 at 9:57 PM, Andreas Sturm <masterstorm123@gmail.com>
> wrote:
>>
>> But the middle primer has one problem, doesn't it?
>>
>> I remember a professor adding TATATAT to each end of the primer, because
>> he said the end may not be correctly synthesized??
>>
>>
>>
>>
>>
>> On Mon, Jan 7, 2013 at 9:53 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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>>>
>>>
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>>
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--
-Nathan

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