Yeah, thank you... Figured that out, should have beed ds-DNA view then I'd have seen it earlier.
Well, AFAIK, the polymerase moves over the DNA strand until - no you're correct. the RBS should have a certain distance from the ATG. However, in chloroplasts that doesn't matter because they seem not to need RBS :D
For pUC19 insertion that may matter. pVIB is based on pBR322, so you've got 20 copies per cell. With pUC19 you'll get 100 copies per cell (IIRC). Immagine that glow ;)
On Wed, Nov 21, 2012 at 8:48 PM, Cathal Garvey <cathalgarvey@gmail.com> wrote:
Hey Nathan, Mega..
Not sure that the frame of the promoter matters so much; it's OK to have
some 5' bits of variable length AFAIK. Rather, the frame of the start
codon (and to a lesser extent the Kozak/Shine-Dalgarno sequence) that
matters.
I'd err on slightly more distance from promoter to start codon than
less, as transcription start points aren't always immediately at the
promoter itself, and may be poorly characterised. Perhaps even variable,
I can't claim to know..
www.indiebiotech.com
On 21/11/12 17:04, Nathan McCorkle wrote:
> Mega, why not just rotate the image in a photoshop-type program, then it
> will be in the normal orientation! It really is that simple though, you're
> just looking at things rotated around! Design your primers so either end
> has a different restriction sequence, then when you PCR the promoter just
> use the same restriction site for the end of the promoter.
>
> Also make sure the ATG of the gene remains in the correct coding frame as
> the promoter, the distance from the start of the promoter to the start of
> the gene should be evenly divisible by 3
>
>
> On Wed, Nov 21, 2012 at 3:42 AM, Mega <masterstorm123@gmail.com> wrote:
>
>> Hi guys,
>>
>> When I do a PCR and then get an operon which is reversly orientated (see
>> picture http://www.ncbi.nlm.nih.gov/gene/3280765 LuxA-G-fre needed) and I
>> need to clone it next to a normally oriented promoter, what can I do to
>> turn it around?
>>
>> I thouht about cutting with restriction enzymes, which will cut it in a
>> way that they just fit together if correctly inserted. But none of the RE
>> have restriction sites that I immagine would fit together...
>>
>>
>> Has anyone experience with this?
>>
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>
>
>
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