Plasmid miniprep kit

[Tom Randall]

I am putting together the reagants for a plasmid miniprep kit for myself and had a simple question if anybody out there has done this. Two of the solutions require guanidium HCl (http://www.roningenetics.org/Protocols.html, about 80% of the way down the page under the heading "Solutions for plasmid minipreps", buffer N3 and buffer PB, 4M and 5M guanidium HCl, respectively). Having always bought commercial kits before, I was wondering if these two solutions really needed autoclaving. I suspect not since guanidium is a strong protein denaturant so it seems highly unlikely anything, including DNases, would survive a 4 or 5M solution of this. I will test my kit without autoclaving first, but any suggestions would be welcome.

[Derek]

I've made my own buffers before. I followed the formulation on http://openwetware.org/wiki/Qiagen_Buffers

 

I believe I filtered those solutions rather than autoclaving since I had a filter already set up. I doubt that either was necessary, though, since as you say the guanidium is at such high concentrations.

 

--Derek

[Cory Tobin]

No autoclaving necessary.

Also, I wrote an article for the next volume of Citizen Science
Quarterly, which should be out shortly I think, that has recipes for
all the miniprep solutions using only household ingredients. I found
that sodium chloride works just fine in place of guanidine
hydrochloride as long as the concentration of salt in the final
mixture (buffer P1 + P2 + N3) is above 2M and the pH is below 5.5. My
homemade recipe for N3 is 11.5g table salt, 3.7g potassium chloride,
and 43mL of distilled white vinegar (no extra water added). You can
get the potassium chloride at the grocery store (I found it at
Ralphs). It's sold by Morton Salt as some sort of additive for water
softener tanks. Anyways, if you give it a try let me know how it
works.

Also, an added benefit of using the NaCl is that very little RNA is
bound to the silica compared to guanidine (which is why kits come with
RNase to add to buffer P1). There is a little RNA that comes along
for the ride, but I imagine if you play around the the pH and salt
concentration you can probably minimize that even more.

-cory

[Derek]

Wow, Cory, that's great! I look forward to reading and trying that.

[Mac Cowell]

Yeah that sounds great. Guanidine hydrochloride is considered toxic waste by most university labs, so a drain safe alternative is great.  

Mac

231.313.9062 // @100ideas // sent from my rotary phone

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[Jeswin]

What do you plan on using for the silica columns? I don't think the
companies (e.g. QIAGEN) don't sell columns outside of kits. Or are you
using a generic silica column, under the assumption that MiniPrep
columns are the same.

[Cory Tobin]

> What do you plan on using for the silica columns? I don't think the
> companies (e.g. QIAGEN) don't sell columns outside of kits. Or are you
> using a generic silica column, under the assumption that MiniPrep
> columns are the same.

You can use diatomaceous earth (sold at pool supply stores). Instead
of packing them into a column and centrifuging the liquid through the
column you add the diatomaceous earth powder to the solution, vortex
to suspend it, then quickly centrifuge to pellet the diatomaceous
earth, then either pour or pipette away the supernatant. As described
here:

Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van
Dillen, P. M., & van Der Noordaa, J. (1990). Rapid and simple method
for purification of nucleic acids. Journal of Clinical Microbiology,
28(3), 495-503. Retrieved from
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=269651&tool=pmcentrez&rendertype=abstract

-cory

[Jeswin]

On Mon, Mar 26, 2012 at 7:21 PM, Cory Tobin <cory....@gmail.com> wrote:
>
> You can use diatomaceous earth (sold at pool supply stores).  Instead
> of packing them into a column and centrifuging the liquid through the
> column you add the diatomaceous earth powder to the solution, vortex
> to suspend it, then quickly centrifuge to pellet the diatomaceous
> earth, then either pour or pipette away the supernatant.  As described
> here:
>
> Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van
> Dillen, P. M., & van Der Noordaa, J. (1990). Rapid and simple method
> for purification of nucleic acids. Journal of Clinical Microbiology,
> 28(3), 495-503. Retrieved from
> http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=269651&tool=pmcentrez&rendertype=abstract
>

Cool. I wonder what the efficiency difference is between this and
commercial columns.

[Tom Randall]

Epoch is a good source.

http://www.epochlifescience.com/Product/SpinColumn/minispin.aspx

They ship to your house. They arent as re-usable as the Qiagen columns, a higher failure rate, but the price is good.

[Cory Tobin]

> Cool. I wonder what the efficiency difference is between this and
> commercial columns.

If I use the diatomaceous earth method but with Qiagen solutions I get
0-10% less DNA than compared to Qiagen solutions + Qiagen columns.
When I use my completely homemade solutions I get 25-50% less DNA than
Qiagen. So it's obviously not perfect but for most DIY folks' needs,
it's probably sufficient.

-cory

[Dakota Hamill]

Epoch is a good source.

http://www.epochlifescience.com/Product/SpinColumn/minispin.aspxThey ship to your house. They arent as re-usable as the Qiagen columns, a higher failure rate, but the price is good.

Thanks for that link Tom, those prices are awesome!  That's 50% cheaper than anywhere else I've found them for the 50 kits.  Sequencing looks cheap too, $5.  I was just about to spend $80 on a Qiagen kit, Epoch is only $30. 

[Nathan McCorkle]

Epoch sent me samples for free... thought they probably can't sustain
doing that much.

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Darrell Montana]

I've successfully used Epoch spin columns for multiple applications, including PCR clean up, removal of restriction enzymes,  gel extraction, and minipreps,  A friend of mine gave me the used columns from his lab (which they don't recycle) and I make all the solutions myself.   They are easy to recycle as well.   When your finished with them, let them soak for 24-48 hrs in 1M HCl, wash 5X with dH20, and 2X with TE, and they should be good to go.  You can also make your own columns if you happen to have any Whatman GF/F paper.   Just remove the glass filter that is in the column, cut out a very small piece to replace it with (I usually scrunch up an ~1.0 X 1.0 cm piece and stuff it in the column with a pipette tip.

[Cory Tobin]

> Cory,
>
> Sorry for the thread necro here. I was wondering if your article came out
> on making buffers from kitchen ingredients. It's relevant to my PhD project
> which is looking at bringing a DIY geomciro/chemical biology lab folks
> traditionally exclude from science.

I don't think so. I'm not sure whatever happened to csq but I don't
think it's being published any more. But you can read the protocol
here http://wiki.biohackers.la/Miniprep or here
https://github.com/cathalgarvey/biohacking-protocols/blob/master/Miniprep%20Using%20Diatomaceous%20Earth.md

-cory

[Dakota Hamill]

you might also find these two links useful.

http://openwetware.org/wiki/Miniprep/Qiagen_kit

and a recipe for all the buffers

http://openwetware.org/wiki/Qiagen_Buffers

[narxl]

Syd Labs sells cheap spin columns for plasmid miniprep. http://www.sydlabs.com/spin-column-for-plasmid-miniprep-p110.htm