Transgenic Core Facility:
DNA
Preparation Protocols
(Click here to download
an MS Word file of these protocols)
DNA Preparation Protocols
DNA purification for transgene
microinjection
- Recombinant plasmid
should be purified by CsCl gradient or Qiagen Maxi-prep. The insert must
then be separated from the vector by restriction digestion, since vector
sequences can significantly alter the expression of transgenes.
- Separate the insert
from the vector on an agarose gel run in Tris/Acetate/EDTA(Not Tris/Borate/EDTA)
buffer. Use 5 mg/ml Ethidium bromide for gel staining. Visualize the DNA
with long wavelength UV light to avoid damaging the ethidium
bromide-intercalated DNA.
- Excise the gel slice
containing the gene fragment of interest and electroelute the DNA, or else
process though Qiaex gel extraction kit (Qiagen, Inc.) according to
manufacturer's instructions.
- Ethanol precipitate the
DNA. For ethanol precipitation of sample, add 1/10 volume of 3 M NaAcetate,
mix, then add 2-2.5 volumes of 100% ethanol. Incubate at -20º C overnight,
then spin 5 minutes in a microcentrifuge to pellet the DNA. Resuspend in
Elutip buffer (Schleicher and Schuell)
- Pass the DNA through an
DEAE Elutip-D mini-column.(Schleicher and Schuell catalog # 27370).
- Ethanol-precipitate the
DNA as above. Wash the pellet several times with 70% ethanol and dry the
pellet under vacuum. The washing and drying steps are very important, as
residual salt and ethanol are lethal to the developing embryo. Resuspend in
sterile MiTE (10mM Tris/0.1mM EDTA, pH 7.4 prepared with Sigma embryo tested
water (Cat # W-1503) or Milli-Q quality water.
- Estimate the DNA
concentration. Run a small sample of the prepared fragment on a agarose gel
alongside uncut plasmid and a set of known standards such as lambda-HindIII
markers A photograph of this gel must accompany all requests for
microinjection. Adjust the DNA concentration to around 4ng/ul in MiTE and
make sure there is at least 150 ul of volume to be submitted to the
Transgenic Core Facility.
Be sure to label the tube with the
concentration and volume and your name for the DNA construct before submitting
the DNA to the Transgenic Core Lab. The DNA should be brought to Room C1732
(Macromolecular Core Facility) no later than noon of the Friday before your
first injection is scheduled. The DNA should be put in Room C1732
(Macromolecular Core Facility) the 2nd freezer on the left, just past the 2nd
door on the left of and should be properly labeled for easy identification.
NOTE - All PAPERWORK required should be
placed in the tray inside the door of Room C1735 (Mass Spec/Proteomics Core
facility), NOT left in C1732
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Last revised:
January 24, 2008