- LMP agarose (Gibco/BRL cat#15517-014)
- GELase (Epicenter Biotechnology cat# G09050)
- Buffer (Epicenter Biotechnology cat# G191ML)
At least 2 µg in 50 µl of a DNA construct should be supplied as
a single linear DNA fragment prepared as described below.
While a variety of other methods are available to prepare DNA, we
recommend that you use the following procedure.
IMPORTANT NOTE: The LMP agarose should be from
Gibco/BRL (cat#15517-014) as some other brands of agarose are toxic
and kill the embryos.
The source of the GELase is Epicenter Biotechnology
(1-800-284-8474; GELase cat# G09050; Buffer cat# G191ML). You may
follow the Epicenter GELase protocol exactly, or use the following
protocol, or use Promega's AgarAce.
- Digest at least 20 µg of DNA with appropriate enzymes to
completely remove vector sequences (total volume of, say, 100 µl,
with 5 µl or so of each enzyme needed). Digest 1 hr to O/N.
Add 40 µl 6X loading buffer (we use a Ficoll based buffer), mix
completely. The exact amount to digest depends upon the resolution
of vector from insert in the gel, gel well volumes, etc. Use
your experience and common sense!
- Run digested DNA on appropriate percentage (usually 0.7-1.0%
for transgenic constructs) LMP agarose gel (Gibco/BRL cat#
15517-014). Load into several individual wells (10 x 20 µl
samples), or in one continuous well (about 1 µg DNA per cm width of
comb). Run gel slowly in 1X TAE at 60 volts for several
- On UV light box, quickly cut out appropriate bands trying to
trim away as much agarose as possible. Keep bands away from UV
light for as long as possible. Minimized exposure to UV
increases cloning efficiency and reduces DNA damage. Put gel
slices into eppendorf tube. Volume should be about 500
µl. If necessary, split bands equally into 2 tubes.
- Place capped tube in 65°C water bath for 10 min, vortex well
(but quickly), reincubate at 65°C for 10 min, vortex and place tube
at 40-42°C for 10 min.
- Add "50X" GELase buffer to melted agarose pieces to make it 1X
- Add 2 µl GELase/500 µl volume. Incubate overnight at
- To check GELase digestion, place tube on ice for 10 min.
If still liquefied, proceed. If there are any indications of
gelling or viscosity, remelt as above (step 4) and repeat GELase
treatment for several hr.
- Perform two phenol extractions with equal volume (about 400 µl)
at RT, of saturated phenol (first interface will have material
deposited; second should not. If it does, do one extra phenol
extraction). Do one phenol:chloroform:isoamyl alcohol extraction
(25:24:1) and one fresh chloroform extraction. NOTE: Even though
there are a lot of extractions here, by careful pipetting and
recentrifuging interface components into the conical tip of the
eppendorf tube, you should still be left with 95% of your solution
(and therefore DNA) at the end of this procedure.
- Add slightly more than 1/10 volume of 3.0 M sodium acetate (pH
5.2), e.g. if you have 400 µl of solution, add 50 µl. Then add two
volumes of 100% EtOH (fill tube, essentially), mix well, let sit at
RT 10 min, and microcentrifuge 15-30 min to recover DNA.
- Rinse pellet with 500 µl 70% EtOH (RT), dry pellets, and
resuspend in TE.NOTE: Increases in purity/quality of DNA are
thought to be achieved if more than one round of 70% EtOH rinsing
is performed, or if two or more EtOH reprecipitations are carried
out (no phenol extractions; just redissolve the DNA in 400 µl TE,
add 1/10 volume sodium acetate, and at least 2 volumes EtOH, then
recover as above).
- Reprecipitate DNA by adding "1/10 volume" 3.0 M sodium acetate
(pH 5.2), and at least 2 volumes of 100% EtOH. Precipitate as
you are used to doing (maybe use dry ice for 15 min, or O/N at
-20°C, and recover by centrifugation - full speed microfuge for
15-30 min). Rinse pellet with 70% EtOH (RT), respin 5 min,
aspirate S/N, wash again with 70% EtOH. Respin, aspirate,
dry. Redissolve in injection buffer (10 mM Tris pH 7.5, 0.1
mM EDTA; NOTE LOW EDTA CONCENTRATION).
- Determine OD260. Run an aliquot on a gel
adjacent to size markers to determine DNA quality; photograph
- Keep at -20°C and bring the DNA fragment to the
Source: TMESCSR 4/10