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Published on: October 14, 2011
Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay
D M Zimmer1, P R Harbach, W B Mattes
1Investigative Toxicology, Pharmacia & Upjohn, Kalamazoo, Michigan 49001, USA.
Abstract:
We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4 degrees C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C. The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation.
Insights
This study optimized the lambda cI/cII transgenic mutation assay for detecting genetic mutations. Key parameters like DNA storage and incubation conditions were evaluated to improve assay reliability and sensitivity.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- The lambda cI/cII transgenic mutation assay is a sensitive method for detecting genetic mutations.
- Optimization of experimental parameters is crucial for accurate and reliable results.
Purpose of the Study:
- To evaluate and refine key experimental parameters of the lambda cI/cII transgenic mutation assay.
- To enhance the assay's sensitivity and reproducibility for mutation detection.
Main Methods:
- Assessed the impact of DNA storage conditions (temperature, duration) on mutant frequency and phage titer.
- Compared phage plating efficiency on different media (LB, TB1) and E. coli strains (G1217, G1225).
- Investigated the effect of incubation temperatures (37°C, 24°C) on mutant detection.
Main Results:
- DNA storage at 4°C for up to 4 months did not affect mutant frequency or titer.
- Phage storage at 4°C for 28 days showed a trend towards higher mutant frequency compared to day 0.
- Optimal phage titer and mutant detection were achieved using the G1217 E. coli strain incubated at 37°C.
Conclusions:
- The lambda cI/cII assay is robust regarding DNA storage.
- Specific plating conditions (G1217 strain, 37°C incubation) significantly enhance mutant detection sensitivity.
- Established optimized parameters improve the reliability of this transgenic mutation assay.
