Ornitz Lab Protocols

 

David M. Ornitz, M.D., Ph.D.

Professor, Department of Developmental Biology

Washington University School of Medicine

 

  • Ornitz Lab Protocols

  • 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...".

  • 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...".

  • 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...".

  • 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...".

  • 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...".

  • 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...".

  • Other Protocols