Murine Models Core Transgene Preparation Protocol
The following procedure applies to small transgenes constructed
in bacterial plasmids. For Bacterial Artificial Chromosomes, purify the
construct with a Qiagen large construct kit and elute in construct buffer
(10mM TRIS, pH=7.4, 0.1mM EDTA). This buffer should be made in Millipore
quality water. Contact Ted Simon for a supply of the buffer. Submit the
construct directly in this buffer. Small transgene constructs prepared in bacterial plasmids
should be freed from vector sequences with a restriction digest resulting
in fragments that are readily separated by agarose gel electrophoresis.
Digest enough material to yield 1µg of construct after purification, assuming
a yield of 50%. Perform agarose gel electrophoresis in TAE or TBE buffer.
Visualize the DNA fragments in the gel after electrophoresis with ethidium
bromide and a short exposure to long wave UV light. Ensure no other
band overlaps with the transgene band, and excise the gel fragment containing
the transgene. Recover DNA from the gel slice utilizing the QIAEX II kit
from Qiagen, EXACTLY following the kit instructions. For fragments larger
than 10Kb, electroelution before QIAEX gives better yields. Elute the DNA from the QIAEX resin with 10mM Tris, pH=8.5
(kit EB buffer). Repeat the elution and combine the two eluants. Precipitate
the DNA with 1/10 volume 3M sodium acetate, pH=5.2, and 2.5 volumes absolute
ethanol. Chill overnight at -20° or for 15 minutes in dry ice/ethanol,
and pellet the DNA by centrifugation at 12000 x g (full speed in a microfuge)
for 15 minutes. Carefully remove the supernatant with a pipet, and add
an equal volume of cold 70% ethanol. Vortex, spin for one minute, and
carefully remove the supernatant with a pipet. Now allow the pellet to
air dry completely. Add 20 µL of TE or transgene buffer and vortex (transgene
buffer is 10mM TRIS, pH=7.4, 0.1mM EDTA). Allow the DNA to solubilize
for at least 15 minutes with occasional vortexing. Use 1µL of the construct
solution to determine the concentration of the DNA with a fluorometer
or low-volume spectrophotometer. Run 200ng of the construct on an agarose
gel with appropriate size markers and visualize with ethidium bromide
staining. A single, sharp band of the appropriate size should be evident.
We will need at least 500ng of DNA for injection, and this should be provided
in a tube labeled with the construct name and DNA concentration. The construct
name should be short and not contain Greek characters, unusual symbols,
subscripts or superscripts. Ted can provide the electroelution procedure
if needed, and a fluorometer is available in his laboratory. If you prefer a different method than the one detailed above
above, please contact Ted Simon advance. |