Primer Purification

[Mega]

Hi everyone,

 

 

I was ordering Primers, and one primer costs around 45€ (around 100 bp). Purification is the major cost with 30€ (instead of standard  37.50€).

 

A professor of mine told me that I could make a PAGE gel myselve, thus one primer synthesis would just be 15€.

 

 

Given we would have all the equipment, would that be feasible? Would it be of high enough quality?

 

 

Best,

Andreas

[Ashley Heath]

Yes, if you can PAGE purify and extract the oligo yourself then it will be perfectly fine to use. Many of our customers here at SIGMA do that, to save the extra up-front costs.

[Andreas Sturm]

Thanks!! 

And is the money (saving) in relation to effort? Will my first PAGE purification product be so fine I can use it without problems? 

[Avery louie]

why are your primers so long?

On Fri, Mar 15, 2013 at 1:00 PM, Andreas Sturm <masters...@gmail.com> wrote:

Thanks!! 

And is the money (saving) in relation to effort? Will my first PAGE purification product be so fine I can use it without problems? 

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[Nathan McCorkle]

he's mentioned before he's adding in promoters and a few extra restriction sites

--
-Nathan

[Andreas Sturm]

Promoter+RBS  or terminator is attached. Also restriction sites. 

 

 2/3 of the price is purification, not bp price.. 

[Jeswin]

On Fri, Mar 15, 2013 at 1:42 PM, Andreas Sturm <masters...@gmail.com> wrote:
> Promoter+RBS or terminator is attached. Also restriction sites.
>
>

This is for PCR, right? What kind of annealing temperatures are you
looking at? Would it have been cheaper to try to join multiple parts
instead of getting such a big primer?

[Andreas Sturm]

51°C / 54 °C... 

You mean like joining three ssDNA primers:

AAAAAA      BBBBBB

       AAAABBBB

like that?? 

Could that work?

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[Andreas Sturm]

I mean the first primer (AAA) binds to the joining primer and the second primer  (BBB) too. 

And the rest should be filled by polymerase and linked by ligase... 

?

[Nathan McCorkle]

seems like you'd have more basepairs to pay for that way, since you
need redundant overlaps in the separate primers/oligos. Unless the
melting temps are too high/low, what problems would using a longer
primer face? Any?

--
-Nathan

[Jeswin]

On Fri, Mar 15, 2013 at 2:24 PM, Andreas Sturm <masters...@gmail.com> wrote:
> 51°C / 54 °C...
>

What are you doing again? I know fluorescent related, chloroplast(?).

[Ashley Heath]

Well, that would be hard to say, depends on you and your colleagues' experience with the PAGE process. If you can, check Short Protocols In Molecular Biology (Ausubel et. al. 1997) Unit 2.11 "Purification of oligonucleotides using denaturing PAGE".

[Andreas Sturm]

Of course there would be more base pairs, but they only make 1/3 of the price so it stays much cheaper. And it wouldn't have full lenght, 10 bp each would be sufficient to bind to the strand I assume.

In this case it would be a trial, to attach  the lux operon to a chloroplast promoter, and then see if agrobacterium can transfect the chloroplasts... Bad thing, there is no yellow fluorescent protein in the Vibrio Fischeri tmplate DNA I have (and it would reqire even another pair of primers)  to shift the light output to yellow thus increasing light yield... 

However,  when the synthetic DNA from Genome Compiler 's kickstarter project arrives, the primers will be overdue anyway... Just to try my "first PCR with own primers"

Hopefully the DNA will be subcloned into pClean by the synthesis company  as I suggested ;)