home made spin columns (was LB vs TB?)

[Song Tan]

I received several requests for more information on the 
home-made spin columns that I mentioned in my posting on 
using TB media for growing plasmid miniprep cultures.  I 
should mention that there have been quite a few previous 
postings dealing with home made spin columns in this 
newsgroup.  Some that I have archived are (in no particular 
order)

1.  Spin columns - revisited
    from Alexander L. Klibanov on 9th June, 1993.
2.  Spin columns       from Mike Poidinger on 21 May 92.
3.  dye removal from ABI terminator reactions  
    from Raj Shankarappa on 19 Nov 92.

These and other previous postings have some neat ideas and 
are worth picking up via Gopher (I will repost them if 
there's a demand).  Spin columns are also mentioned at the 
end of Paul Hengen's nice FAQ for this newsgroup available 
by anonymous ftp from  ncifcrf.gov as the file 
pub/methods/FAQlist and from net.bio.net as 
pub/BIOSCI/METHDS-REAGNTS/METHODS.FAQ.

O.K., for my spin columns (which I learnt from my boss, Tim 
Richmond):  it's basically a blue pipette tip spun in a 5 ml 
polypropylene tube (Nunc-type tube).  We use the top two 
thirds of a 1.5 ml Eppendorf tube (lid snipped off, and then 
cut just below the taper) to provide the holder for the blue 
tip.  This holder can be reused.  In more detail:  take a 
Gilson blue tip, plug the bottom with some siliconized glass 
wool.  Fit the holder on top of a 5 ml polypropylene tube (I 
use inexpensive non-sterile versions instead of the 
expensive genuine Nunc tubes).  The plugged Gilson blue tip 
will fit nicely into the holder (genuine Gilson blue tips do 
fit, some other blue tips I've tried don't fit).  Fill the 
blue tip with your favorite gel resin.  Centrifuge the spin 
column assembly in a table top centrifuge, say, 1000 rpm for 
5 minutes.  Discard this first eluent at the bottom of the 
polypropylene tube.  Now add your sample to resin, 
centrifuge again at the same speed and for the same amount 
of time, and then collect your sample from the 
polypropylene tube.

The holder I described above can be a little troublesome to 
make (easy to cut yourself if you use a razor blade), but 
since it's reusable, you can make a bunch and never have to 
worry about them again.

I use Sepharose CL-6B or Sephacryl S400, both equilibrated 
in TE(10, 0.1), as the resin for removing digested RNA from 
my minipreps.  500  l of packed Sepharose CL-6B (i.e. the 
volume after the first spin) is enough to remove RNA (at 
least so that it's not visible on an ethidium bromide 
stained PAGE gel) from a 5 ml plasmid miniprep, but please 
remember that I am using the modification of Feliciello and 
Chinali to remove some RNA during the alkaline lysis.  An 
aside:  Pharmacia markets prepacked spin columns containing 
S400 or Sepharose CL-6B for US$3 to 5 each (Swiss prices 
converted to US$).  I estimate the homemade version to be 
one tenth as expensive.  

Some people have also asked about using spin columns to 
remove unincorporated nucleotides after labelling or for 
removing primers after PCR.  I do use the same spin column 
assembly to remove unincorporated nucleotides after 
kinasing, but then I use Sephadex G25 Fine as the resin.  
Cross-linked Sepharose and Sephacryl should both be usable 
for removing primer oligos after PCR, but it does depend on 
the size of your PCR product.  The cutoff for Sephacryl S200 
appears to be around 100 bp (Pharmacia quotes exclusion 
limits of 118 bp for S200 and S300, 271 bp for S400 and 194 
bp for Sepharose CL-6B).  I have use S400 spin columns to 
purify PCR products, but I haven't used this material for 
any applications that critically depended on the removal of 
the primers.

Although I have used the spin column assembly described 
above for years now and it works perfectly well, I would 
prefer an even simpler setup.  If I can, I will move over to 
the spin column made of one 0.5 ml Eppendorf tube with a 
hole pierced through the bottom, placed inside one 1.5 ml 
Eppendorf tube as described by several in this newsgroup.  
Th resin is pipetted over glass beads or glass wool in the 
small tube.  I had problems using this setup when I was 
using Sephacryl S400 HR resin because that resin kept 
seeping through the glass beads.  (I know, I know, I 
shouldn't have used the expensive and smaller HR resin for 
spin columns, but we had bottles of the stuff lying around.)  
I haven't actually tried that setup with Sepharose CL-6B 
resin, but others have described their successes of doing 
exactly this.  

I hope that you'll find this information useful.  I've got a 
lot of useful hints and tips from the net in the past few 
years and it's nice to be able to be give something back.  

Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich
email:  tan at aeolus.vmsmail.ethz.ch

Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich
email:  tan at aeolus.vmsmail.ethz.ch