CloneChecker™ System is a rapid and efficient method for screening recombinant bacterial colonies for the presence of target plasmid DNA. CloneChecker™ System contains reagents for analysis of plasmid DNA
directly from a bacterial colony or from liquid culture. Partially purified plasmid DNA obtained by using CloneChecker™ System reagents can be analyzed by agarose gel electrophoresis and verified by direct size comparison, restriction enzyme analysis, or PCR analysis.
CloneChecker ™ System contains sufficient materials for the analysis of 100 bacterial colonies by Restriction Enzyme Analysis and 100 bacterial colonies by Supercoiled DNA Analysis.
Green Solution 800 μL
Yellow Solution 500 μL
Red Solution 500 μL
Loading Buffer 600 μL
Note: Composition of reagents is proprietary and non-hazardous.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
After overnight culture (16–20 h) of plated transformation reaction:
- Pick a bacterial colony (1 to 2 mm in diameter) with a pipette tip and suspend it in 6 μL of fresh growth media (e.g., Luria Broth). Mix thoroughly. Note: Ensure that the entire colony is picked, while avoiding agar. Agar may cause inhibition of the reaction.
- Transfer 3 μL of suspended colony to 8 μL of Green Solution. Mix by pipetting up and down 3 times. Note: The remaining 3 μL of suspended colony can be used to inoculate an overnight culture for further analysis.
- Heat the sample at 100°C for 30 s in a boiling water bath or thermal cycler.
- After sample cools to room temperature, add 2 μL of digestion mix containing 1 μL of 10X reaction buffer and 1 μL of restriction enzyme (1–10 units per reaction).
- Incubate sample for 30 min at the appropriate temperature for the restriction enzyme used. When using 1 unit o enzyme, incubate for 30 min. When using 10 units of enzyme, incubate for 10 min.
- Add 2 μL of Blue Loading Buffer to sample. Mix and load into wells of an agarose gel. After electrophoresis, visualize DNA by staining gel in 0.2 μg/mL ethidium bromide for 10 min.
MAN0000993 Rev. date: 20-May-2010