magnetic M13 preps
rick at GENEMAN.WUSTL.EDU
Wed Mar 3 08:36:53 EST 1993
finabo.abo.fi (Jarmo Niemi) asks:
>I am doing sequencing from M13 templates, and would appreciate if you could
>give me a reference or some other pointers to the ssDNA prep method
>using magnetic beads.
You have a couple of choices: 1) use non-derivatized magnetic particles, or
2) use streptavidin-coated magnetic particles.
Amersham sells a kit which contains non-derivatized particles; the idea is that
the particles are trapped in an aggregate of phage particles (when you add PEG)
or an aggregate of DNA (when you add ethanol). Alderton et al. (Anal. Biochem.
201, 166) have described using this method in 96-deep well plates. If the
particles are purchased from Amersham, cost per template is about $1-1.50 US.
However, you can substitute non-derivatized particles from Bang's Laboratories
(Carmel, IN) and knock the cost down to about $0.07 per template (the catalog
number for the right particles is M0008008CN). The method works well, although
is a bit tricky for an inexperienced technician.
Alternatively, streptavidin-coated beads can be used. The trick here is to
couple a biotin-labeled oligonucleotide with homology to the M13 sequence to
the beads. After phage proteins are disrupted with SDS & heat, the bead-oligo
is hybridized to template DNA. After a few washes, the M13 DNA is eluted by
heating. Hawkins (DNA Sequence 3, 65) has described the use of this method
with 96-well plates.
The advantage of the second method is DNA quality - you're using an affinity
purification method with plenty of washing. The disadvantage is cost. The
SA-coated beads are more expensive ($0.70 - 1.00 US per template) than the
non-derivatized particles. However, the beads may be recovered and washed
and used again to help reduce cost.
Hope this helps!
More information about the Methods