Re: Get a plasmide - buy? share?

Hi Ethan!

Great to hear you're getting into transformations. I hope you aren't
waiting too long until your first results.

As to "Rolling your own" minipreps, you actually don't need columns at
all. Indeed, I am told anecdotally that purifying without columns gives
far greater yields!

Instead of using Columns, you can purify without by precipitating the
plasmid DNA using alcohol, centrifuging the DNA to the bottom of an
eppendorf tube after precipitation, and washing by resuspending in an
alcohol-based wash buffer followed by another resuspension.

Look at it this way; the columns simply offer a nice place for the DNA
to precipitate, and hold onto the precipitated DNA while you wash it.
They "release" DNA when you rinse them with a buffer that can
successfully dissolve it away. You don't need this nice place, if you
just use centrifugation to keep the DNA where you can handle it while
removing buffers.

The hard part without a column is drying the DNA free of alcohol, but
not *too* dry (because it's harder to redissolve), and then dissolving
it again. If it's been dried for not-long-enough, it will have residual
alcohol in it which might affect freezing, reactions, or even its
ability to dissolve. If it's too dry, you might need to leave it
overnight to redissolve correctly!

In any case, you can get columns and miniprep kits, along with a bunch
of other stuff, from NBSbio.co.uk if you live in Europe. Just make sure
to *order by email*, and tell them that you are *not* VAT registered.
They've always shipped to me without hassle, and are very friendly and
professional.

On transformations and enzymes; it massively increases transformation
efficiency if your cells have no restriction/modification systems, but
it won't entirely prevent it. My favourite culture, B.subtilis 168, has
an enzyme that cuts similarly to XhoI; if there are any XhoI sites
present, your efficiencies are 100 times lower. Once transformed though,
the methylase kicks in and the DNA is stable, and can be transformed
back in after a miniprep without problems (but not after PCR, because
PCR doesn't methylate!).

For difficult transformations, many companies offer methylases to
protect the DNA before transformations or reactions. I've never used
them, and I imagine they are a pain in the arse to use.

If you *are* in Europe, we'll have to meet sometime at a summit to talk
transformations! If not, we'll surely get a chance anyway eventually. :)

On 22/12/11 01:45, Ethan wrote:
> First off, greetings! I have been lurking for a bit, but this is my
> first post here.
>
> Thank you all for the valuable information in this thread. I am still
> trying to get my lab set up to do basic transformations. One thing I
> am curious about is purification of plasmids so that I can continue to
> use the plasmids that I have. Looking for miniprep columns online, I
> am unable to locate a seller that ships to residential addresses. Is
> there a way to circumvent that? Also, specifically to Cathal Garvey,
> you mentioned that you could "roll your own" in the context of
> columns. Do you have any more information on this? I would like to
> keep costs to a minimum, so making my own equipment is definitely of
> interest to me.
>
> Additionally, I have a general question about transformations (I have
> done a few before, but I didn't have full details on the strains of
> bacteria at the time). When performing a transformation, is it
> necessary to work with a strain that is deficient in restriction
> modification, or is that only an issue if you are working with a fully
> synthetic sequence (i.e. just purchased from a DNA synthesis company)?
> Thank you, everyone!

--
www.indiebiotech.com
twitter.com/onetruecathal
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PGP Public Key: http://bit.ly/CathalGKey

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