A. Protocol for making electrocompetent bacteria cells
Note: This is a generic protocol for making electrocompetent bacterial cells.

1) Use a fresh colony or frozen stock of  DH10B cells to inoculate 10 ml of LB medium in a 50 ml tube (for BJ5183 cells, to inoculate 10ml LB containing 30 ug/ml streptomycin).  Grow cells in a shaker overnight at 37^oC.

2) Dilute 1 ml of cells into 1000 ml of LB medium (for BJ5183, use streptomycin-containing LB medium) in eight 1 liter flasks (125 ml each).  Grow for 4 to 5 hour with vigorous aeration at 37^oC,  until A₅₅₀ is ~0.8.

3) Collect cells in four 250 ml conical centrifuge tubes and incubate on ice for 10 minutes to 1 hour. (The longer the cells are incubated the higher the competency.)

4) Pellet cells by centrifuging at 3,000 rpm (2,600 g) at 4^oC for 10 min.

5) Wash the cell pellet by resuspending in 1000 ml of sterilized, ice-cold WB (WB = 10% ultra pure glycerol, 90% distilled water, v/v)

6) Centrifuge the cell suspension at 3,000 rpm (2500 g) for 30 min.

7) Repeat steps 5 and 6.

8) Pour off the supernate roughly, gently pipet most of supernate off, leaving about 20 ml, transfer the cell suspension to a 50 ml tube.  Spin at 3,000 rpm x 10 min, and pipet all but 5 ml of the supernate out (for BJ5183 cells, the final total volume should be limited to 2-3 mls).

9) Resuspend cell pellet in the WB remaining in the tube.  Aliquot 20 ul per tube (the tubes should be pre-chilled at  -80°C) and store the aliquots at -80°C.

B. Checking Competency of  Electrocompetent Cells

Purified plasmid DNA (10 ng/ul)      1 µl
DH10B competent cells           20 µl

1) Transform cells with Bio-Rad gene pulser (200 Ohms/25µF/ 2.5 KV).
2) Add 1 ml of L-Broth at room temperature.
3) Shake cells at 37^oC for 1 hour in 50 ml tube.
4) Make 100-fold and 1000-fold serial dilutions of the cells in L-Broth.
5) Plate 100 ul of diluted cells on L-agar with 100 ug/ml ampiciliin.
6) Incubate overnight @ 37°C.  Titer should be >10⁸ colonies/ug, and we often achieve 10⁹ colonies/ug.

C. Plaque Assay for Purification and Titration of rAdenoviruses
Note:  this only needs to be done for adenoviruses without GFP marker.  In those which include GFP marker, titration of infectious units (i.e., those resulting in expression of GFP) is simply determined by fluorescence microscopy of your favorite cell type.

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