I am trying to perform complementation studies with Staph but I am having trouble making competent cells of my knock out strain. I think this might be due to the fact that this knock-out strain is a small colony variant. Does anyone have any experience working with small colony variants that can provide some insight?
Hi Uzmal,
The protocol I currently use is below:
Generating Electrocompetent S. aureus.
· Grow O.N. culture of S. aureus in TSB.
· Start 100 mL daily culture (1:100 dilution) in 500 mL flask.
· Grow cells to early exponential phase ( for small colony variants, this is about OD600 = 0.15- 0.2
· Harvest using centrifugation (4,000 x g, 10 min, 4˚C)
· Resuspend cells in 40 mL 10% sterile glycerol solution using vortex.
· Centrifuge (4,000 x g, 15 min, 4˚C).
· Remove supernatant and repeat 10% glycerol wash and centrifuge x2 for a total of 3 washes.
· After final centrifuge, resuspend S. aureus in ~1mL 10% glycerol
· Aliquot into 50 μL samples and store at -80 ˚C.
Thanks in advance!
Always rock the 100ml culture flask covered in ice for say 20-30 mins to achieve uniform and complete cooling of the culture before harvesting the cells; keep cells always on ice. After preparation of electrocompetent cells -my transformation efficiency tends to be high with cells kept in -80 oC overnight
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