F- φ80 lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK-, mK+) phoA supE44 thi-1 gyrA96 relA1 tonA
• High transformation efficiency: >108 cfu/μg (pUC19 DNA).
• Resistance to T1 and T5 phage.
• In vivo digestion of methylated DNA, suitable for site-directed mutagenesis.
•Equilibrate a water bath to 42oC.
•Warm a vial of SOC medium or LB medium to room temperature. Warm selective plates at 37oC for 30 minutes.
•Thaw a vial of 50 μl DMT Chemically Competent Cell on ice, add target DNA (1 to 5 μl ) into the tube as soon as the last bit of ice in the tube disappeared and mix gently. Do not mix by pipetting up and down. Incubate the cells on ice for 30 minutes.
•Heat-shock the cells for 45 seconds at 42oC without shaking. Immediately transfer the tube to ice. Incubate on ice for 2 minutes without shaking.
•Add 500 μl of prewarmed SOC medium or LB medium (without antibiotic) into the tube. Cap the tube tightly and shake the tube at 37oC for 1 hour at 200 rpm.
•Spread 20 to 200 μl from each transformation vial on prewarmed selective plate. The remaining can be stored at 4oC and plated the next day if needed.
•Invert the plate and incubate at 37oC overnight.
•Select colonies and analyze by restriction enzyme digestion, PCR, or sequencing.
•Higher efficiency transformation can be achieved by transforming cells immediately following thawing.
•Avoid repeated thawing.
•Gentle handling is required for the entire procedure.
|Citations & references|
|Characterization, expression and immune activity|