[BBF Standards] question on PoPS devices

[Kim de Mora]

Deepak,

  You need one selectable marker for each piece of DNA you insert into your
organism.  In yeast, the four sikorski vectors exist so that you can use
auxotrophic selection to insert up to 4 different constructs.  If you are
making a strain that uses all four auxotrophies, you need to use drug
resistance selection if you plan to do any further modifications, so doing
as much as you can on a single piece of DNA is advantageous.

  In terms of size, Qiagen miniprep kits will yeild DNA up to about 10 K and
you can use them for larger fragments.  I have a colleague who has done 20 K
fragments successfully, although with a lower yield.

  Assembling large pieces of DNA is not too difficult if you do every step
in parallel.  If the devices are already assembled, then further assembly is
just another ligation and transformation step.  If the devices are not
assembled, the process takes more time but the number of parts you can
assemble together in parallel tends to be dependent on how many minipreps
you are confident doing at once.

  As for the leakiness of the terminator, I think we need an assay or a
standardized measurement technique that could be used to assess this.  It
would be good to know as I think people have reported problems using too
many copies of the strongest terminator in the registry.  Cheers,

Kim



>
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> The table also gives the part number for additional output sections.
>
> Randy Rettberg
>
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