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Table 3 Summary of protocol for high-level electrotransformation of C. pasteurianum and comparison to initial low-level protocol

From: Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum

Electrotransformation parameter Low-level protocol High-level protocol
Cell growth   
 Growth additive None 1.25% glycine (at OD600 0.3-0.4)
 Osmotic stabilizer None Hypertonic sucrose (0.4 M; at OD600 0.3-0.4)
 OD600 at time of harvest OD600 0.6-0.8 OD600 0.6-0.8
Washing and pulse delivery   
 Osmotic stabilizer Isotonic sucrose (0.27 M) Isotonic sucrose (0.27 M)
 Cell membrane solubilizer None 5% (v/v) ethanol 5 min prior to pulse
 DNA amount 5 μg 0.5 μg
 Pulse parameters 2.0-2.5 kV; 25 μF; 200–800 Ω; 6–9 ms 1.8 kV; 25 μF; ∞ Ω; 12–14 ms
Outgrowth   
 Osmotic stabilizer None Hypotonic sucrose (0.2 M)
 Recovery time 16 h 4-6 h
 Transformation efficiency 2.4 × 101 transformants μg-1 pMTL85141 Up to 7.5 × 104 transformants μg-1 pMTL85141
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