EXPERIMENT PROTOCOL

I. Protocol for electroporation of both coagulase-positive and -negative Staphylococci
A. Competent cell preparation
1. Culture Staphylococcus in 20ml B2low* broth with agitation at 37Ž overnight.
2. Transfer all pre-culture into fresh pre-warmed 100ml B2low broth.
3. Culture for 1 hour at 37Ž.
4. Transfer culture broth into two pre-chilled 50ml centrifuge tubes and centrifuge them under x6,000g for 10 min at 4Ž
5. Discard supernatant and re-suspend pellets with cold 30ml (15ml/tube) 1.5M NaCl# (wt/vol, autoclaved).
6. Centrifuge x6,000g for 10 min at 4Ž.
7. Discard supernatant and re-suspend pellets with cold 1ml 10% glycerol (wt/vol, autoclaved).
8. Transfer suspension into new 1.5ml tubes and centrifuge under x5,000g for 5 min at 4Ž.
9. Discard supernatant and re-suspend pellets with cold 1ml 10% glycerol.
10.Measure electronic resistance of suspension with ELEPO21.
11. Repeat step 9-10 until electronic resistance of suspension exceed 6kƒΆ.
12.Aliquot 300ƒΚl in each tube and store -80Ž.

B. Electroporation
1. Thaw the competent cells on the ice.
2. Incubate competent cells at 25Ž for 5min$
3. Centrifuge under x15,000g for 1 min at room temperature.
4. Discard supernatant and re-suspend with 300ƒΚl electroporation buffer**.
5. Mix 19ƒΚl competent cell and 1ƒΚl DNA extracted from B strain.
6. Incubate the mixture at 25Ž for 10min
7. Transfer the mixture into 1mm curvet (room temperature).
8. Elctroporation. (PP: 1800V, 2.5 msec, 50 msec, 1 time, +. TP: 100V, 99msec, 50msec, 5 times, +/-)
9. Recover the reaction mixture with 500ƒΚl mB2 broth*** at 30Ž for 5 hours
10. Spread the bacteria on TSA with antibiotic(s)

*: 25g yeast extract, 10g trypton, 10g NaCl, 5g glucose, 1g K2HPO4 in 1ℓ (wt/vol, pH 7.5, adjust with NaOH). Autoclaved at 121Ž for 15min.
**0.5M sucrose and 10% glycerol in purified water (wt/vol, autoclaved at 121Ž for 15min., filtration is not recommended due to frequent arching).
***: 25g yeast extract, 10g trypton, 25g NaCl, 5g glucose, 1g K2HPO4 in 1ℓ (wt/vol, pH 7.5, adjust with NaOH). Autoclaved at 121Ž for 15min.
#: Low salt buffer is also applicable for low biofilm producing strains
$: Option (Heat inactivation), after 25Žincubation, 56Ž 1min¨cooling on ice 1min. Following steps is the same.
(Heat inactivation of restriction enzymes, referred to Cui et al. 2015. and modified in this study)
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