Bio-Rad Gene Pulser Xcell™ Electroporation Systems User Manual
Page 61
4.
From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the Bacterial
Protocol screen (press 4, then Enter twice). To select the Protocol Detail screen for S. aureus, press
6 or the Down Arrow key to highlight S. aureus, then press Enter. See Section 3.4 for operating
instructions.
5.
Transfer the mixture of cells and DNA to a 0.2 cm electroporation cuvette at room temperature and
tap the suspension to the bottom. Place the cuvette in the ShockPod. Push the chamber lid down
to close.
6.
Pulse once.
7.
Remove the cuvette from the chamber and immediately add 1 ml of SMMP medium containing a
subinhibitory concentration of antibiotic; gently transfer the cells to a sterile 17 x 100 mm tube using
a Pasteur pipette. Incubate 1 hr at 37°C, shaking at 250 rpm.
8.
Check and record the pulse parameters. The time constant should be close to 2.5 milliseconds. The
voltage should be approximately 2.9 kV.
9.
Plate aliquots of the electroporated cells on trypticase soy agar containing selective antibiotic.
Incubate plates for 36–48 hrs at 37°C.
6.2.3 Solutions and Reagents
1.
Trypticase soy agar (TSA): dissolve 40 g trypticase soy agar (Becton Dickinson, Sparks, MD) in 1 L
of water. Autoclave.
2.
B2 medium: 10 g casein hydrolysate (casamino acids), 25 g yeast extract, 5 g glucose, 25 g
NaCl, 1 g K2HPO4; dissolve in 900 ml of water and adjust pH to 7.5; bring volume to 1.0 L.
Autoclave.
3.
0.2 M sodium hydrogen maleate: either 11.6 g maleic anhydride and 4 g NaOH dissolved in 500
ml of water, or 13.8 g maleic acid, 4 g NaOH dissolved in 500 ml water. Autoclave.
4.
2X SMM: 25 ml of 0.2 M sodium hydrogen maleate, 40 ml of 0.1 N NaOH; adjust the pH to 6.5. Add
5 ml of 1 M MgCl
2
, 42.7 g sucrose; dissolve and bring volume to 125 ml. Filter sterilize.
5.
4X Penassay broth: 17.5 g Antibiotic Medium 3 (Becton Dickinson) dissolved in 250 ml of water.
Autoclave.
6.
SMMP: 55 ml of 2X SMM, 40 ml of 4X Penassay broth, 5 ml of 10% (w/v) bovine albumin; adjust
pH to 7.0; filter sterilize. For selection of antibiotic-resistant transformants, add a sub-inhibitory
concentration of antibiotic.
7.
10% (v/v) Glycerol: 12.6 g glycerol (density = 1.26 g/cc) in 90 ml of water. Autoclave or filter sterilize.
55