Calcium Phosphate Transfection of Neurons in Primary Culture-磷酸钙法转染原代培

Developed by Hank Dudek and Zhengui Xia (11/96)
Modified by Steve Finkbeiner (rev. 7/97)
Departments of Neurology and Physiology, UCSF/GIND
415-695-3868; sfinkbeiner@gladstone.ucsf.edu

1. Protocol
   1. make calcium phosphate/DNA precipitate:
      1. variables
         • volume of ppt 24 well: 20-40ul
           60mm plate: 120-200ul
           
         • amount of DNA : 24 well: 2-4ug (eg., for immunostaining)
           60mm: 2-5ug (eg., for RNAase protection)
           
      2. recipe: eg., for 300ul precipitate (scale up or down accordingly):
         1. in a 15ml polystyrene pop-cap tube, mix DNA and CaCl2 (1/2 final volume, therefore 150ul in this case) 15ul 2.5M CaCl2 (1/10 of DNA/CaCl2 volume)
            DNA (eg., 30ug = enough for 10 wells at 30ul ppt per well)
            sterile H2O to 150ul

             

         2. aliquot 2X Hepes-Buffered Saline (HeBS) to second tube (150ul, in this case)
         3. add DNA/CaCl2 to 2X HeBS dropwise with pipetman, while swirling 2X HeBS
         4. let ppt for