An Assay to Study Intra-Chromosomal Deletions in Yeast
2. Experimental Design, Methods and Materials
2.1. Spontaneous Break Recombination Protocol
- Streak cells onto EMM-Histidine plates from the −70 °C freezer. Grow at 32 °C for 3–4 days until colonies appear. Although the cassette is quite stable, to ensure that starting cells are ura−his+, it may be necessary to replica plate the EMM-His onto 5-FOA and choose only those colonies that grow on both plates.
- Resuspend 10 colonies each in 100 μL water in microtubes, count cells and release in 4 mL liquid EMM+UraHisLeuAde at 100 cells/microliter. Incubate tubes at 32 °C in the rotator for approximately 48 h.
- Determine the concentration of the cells in the tubes by counting cells using a hemocytometer and plate onto EMM-Uracil+Phloxin B at 105–106 cells per plate. Because ura− cells tend to cannibalize themselves, sometimes false positives appear. The addition of Phloxin B makes it easier to identify false positive because it stains ura− cells bright red. Phloxin B does not have an effect on recombination rate (Supplementary Tables S2 and S3). Furthermore, we recommend using large 150 mm × 15 mm plates particularly when plating at higher density. Plate a YES control as well for each colony plated on EMM-Uracil at 1000 cells per plate. This control is important to check for cell viability and accuracy in counting. Although we used YES for this control, a better control may be EMM+Uracil+Histidine. This maintains consistency with the experimental plate which is EMM not YES.
- Incubate all plates at 32 °C until colonies appear—usually 3%#x2013;5 days for WT and longer for mutants.
- Count colonies on both the YES control and EMM-Uracil plates and record the numbers. Although Phloxin B allows for easier differentiation of ura4+ prototrophic colonies, to ensure that all colonies on the EMM-Uracil plates are in fact Ura+, this plate can be replica plated onto 5-FOA. All ura4+ colonies that grow on EMM-Uracil should die on 5-FOA.
2.2. Induced Break Recombination Protocol
- Streak cells onto EMM-Leucine+Thiamine plates from the −70°C freezer. Incubate at 32 °C for 3–4 days.
- Resuspend 10 colonies in water, count cells and release in 4 mL liquid EMM-Leucine at 100 cells/microliter. Incubate tubes at 32 °C with rotation for approximately 48 h.
- Determine the concentration of the cells in the tubes and plate onto EMM-Uracil (100 mm × 15 mm plates) at 104 cells per plate. Plate on YES as well at 103 cells per plate.
- Incubate all plates at 32 °C until colonies appear.
- Count colonies on both YES and EMM-Uracil plates and record the numbers.
2.3. Characterization of the Assay
2.5. PCR Analysis
2.6. Data Analysis
3. Genetic Validation of the Assay and Discussion
3.1. Analysis of Spontaneous Breaks
3.2. Analysis of Induced Breaks
Conflicts of Interest
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Lucas, B.E.; McPherson, M.T.; Hawk, T.M.; Wilson, L.N.; Kroh, J.M.; Hickman, K.G.; Fitzgerald, S.R.; Disbennett, W.M.; Rollins, P.D.; Hylton, H.M.; et al. An Assay to Study Intra-Chromosomal Deletions in Yeast. Methods Protoc. 2019, 2, 74. https://doi.org/10.3390/mps2030074
Lucas BE, McPherson MT, Hawk TM, Wilson LN, Kroh JM, Hickman KG, Fitzgerald SR, Disbennett WM, Rollins PD, Hylton HM, et al. An Assay to Study Intra-Chromosomal Deletions in Yeast. Methods and Protocols. 2019; 2(3):74. https://doi.org/10.3390/mps2030074Chicago/Turabian Style
Lucas, Bailey E., Matthew T. McPherson, Tila M. Hawk, Lexia N. Wilson, Jacob M. Kroh, Kyle G. Hickman, Sean R. Fitzgerald, W. Miguel Disbennett, P. Daniel Rollins, Hannah M. Hylton, and et al. 2019. "An Assay to Study Intra-Chromosomal Deletions in Yeast" Methods and Protocols 2, no. 3: 74. https://doi.org/10.3390/mps2030074