TransDB3.1 Chemically Competent Cell is specifically designed for chemical transformation of DNA. This cell contains the gyrA462 gene which provides resistance to the toxic effects from the ccdB gene. TransDB3.1 Chemically Competent Cell can be used for transformation and propagation of plasmid containing the ccdB gene. It permits a transformation efficiency of over 108 cfu/μg DNA (tested by pUC19 plasmid DNA).
F- gyrA462 endA1 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB-) supE44ara-14 galK2 lacY1 proA2 rpsL20(SmR) xyl-5 λ- leu mtl1
• High transformation efficiency: >108 cfu/μg (pUC19 DNA).
• Transformation and propagation of plasmids containing the ccdB gene.
•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 100 μl of TransDB3.1 Chemically Competent Cell on ice, aliquot 50 μl of the cells into a prechilled 1.5 ml tube, add target DNA (1 to 5 μl ) into the tube. 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, mix well and shake at 37oC for 1 hour at 200 rpm for cell recovery and for the expression of antibiotic resistance.
•Spread 20 to 200 μl from each transformation vial on a 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.