| Panning protocol, based on Wysocki
& Sato, PNAS 75, 2844-2848. Antibody-coated dishes We use bacteriological 60mm plates, Falcon 1007 or equivalent, or 10cm dishes such as Fisher 8-757-12; we use Sheep anti-mouse affinity purified antibody from Cooper BioMedical (Cappell) and dilute it to 10 micrograms per ml in 50mM Tris HCl, pH 9.5. Add 3ml per 6cm dish, or 10ml per 10cm dish. Let sit ca. 1.5 hrs, remove to next dish 1.5hrs, then to 3rd dish. Wash plates 3x with 0.15M NaCl (a wash bottle is convenient for this), incubate with 3ml 1mg/ml BSA in PBS overnight, aspirate and freeze. Panning (cells transfected by protoplast fusion) Cells will be in 60mm dishes. Aspirate medium from dish, add 2ml PBS/0.5mM EDTA/0.02% azide and incubate dishes at 37 for 30 min to detach cells from dish. Triturate cells vigorously with short pasteur pipet, and collect cells from each dish in a centrifuge tube. Spin 4 min setting 4 (200xg). (takes 5 min) Resuspend cells in 0.5 -1.0 ml PBS/EDTA/azide/5% FBS and add antibodies. Incubate >30 min on ice. Add = vol. PBS/EDTA/azide, layer carefully on 3 ml PBS/EDTA/azide/2% Ficoll, spin 4 min setting 4. Aspirate supernatant in one smooth movement. Take up cells in 0.5ml PBS/EDTA/azide and add aliquots to antibody coated dishes containing 3ml PBS/EDTA/azide/5% FBS by pipetting through 100 micron Nylon mesh (Tetko). Add cells from at most two 60mm dishes to one 60mm antibody coated plate. Let sit at room temperature 1-3 hours. Remove excess cells not adhering to dish by gentle washing with PBS/5% Serum or with medium. 2 or 3 washes of 3ml are usually sufficient Plasmid Recovery From Selected Cells Add 0.4 ml 1% SDS, 10 mM EDTA to plate. Let sit 20 minutes (can be as little as 1 min. if there are practically no cells on the plate). Pipet viscous mixture into microfuge tube. Add 0.1ml 2.5M KOAc, mix, put on ice 20 minutes. Spin 4 min, remove supernatant carefully, phenol extract (twice if the first interface is not clean), add 10 micrograms linear polyacrylamide (or other carrier), fill tube to top with EtOH, precipitate, and resuspend in 0.1 ml. Add 3 vol. EtOH/NaOAc, reprecipitate and resuspend in 0.1 ml. Transform into MC1061 or equivalently competent bacterial strain by electroporation. |