Ornitz Lab Protocols
David M. Ornitz, M.D., Ph.D.
Professor,
Department of Developmental Biology
Washington University School of
Medicine
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Alkaline Phosphatase Assay, Alkaline Plasmid Prep:
"...heat 200 µl sample to 65¡C 5-10 min. to kill endogenous Alk
Phos. Ctg. 15 sec. Mix 100 µl sample + 100µl 2X Seap buffer
measure OD 405 in Kinetic plate reader or dilute with 800 µl H2O
and read OD 405...".
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BaF3 Cell Methods:
"...BaF3 cells and transfected clones are washed twice with RPMI
media lacking IL3. 2.2x104 cells are plated per well in 96 well
microtiter plates. Appropriate growth factors (0-2nM) and
heparin (0-5µg/ml) are added in a total volume of 200µl...".
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BrdU Immunohistochemistry:
"...This
method utilizes formalin-fixation, paraffin-embedding, and an
anti-BrDU mAb that detects BrDU in ssDNA after denaturation and
neutralization. Because it has been optimized for midgestational
murine lens analysis, it may require further adjustment for
other tissues...".
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LacZ Cell Staining, LacZ Embryo Staining:
"...Embryos are first dissected away from the placental material
and the amnion is removed. When dissecting many embryos at once,
the initial embryos can be stored in 1XPBS while other embryos
are dissected...".
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FGFR-AP-FGF Binding Assay, PCR Assays for Knockout Mice, RNase
Protection Assay:
"...The bound receptor was quantified by measuring the initial
rate of substrate hydrolysis at 405 nm and same amount of
proteins was used in the binding assays...".
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Tail Prep:
"...Avoid Shearing of the DNA by limiting mixing to inversion
and gentle shaking. Vortexing of samples is not required to
perform the following protocols. Avoid contamination by
rigorously cleaning pipetman and using autoclaved tips during
manipulation of genomic DNA and reagents to be used with genomic
DNA...".
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