SBB10AssayTeam4-Notes: Difference between revisions
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== General Procedure == | == General Procedure == | ||
Part 1 <br> | |||
Experiment: <br> | Experiment: <br> | ||
1. Cotransform cells with plasmid containing the tos site and N15 protelomerase expressing plasmid. <br> | 1. Cotransform cells with plasmid containing the tos site and N15 protelomerase expressing plasmid. <br> | ||
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5. Digest the resulting plasmid with ''a restriction enzyme''. <br> | 5. Digest the resulting plasmid with ''a restriction enzyme''. <br> | ||
6. Analytical Mapping <br> | 6. Analytical Mapping <br> | ||
Controls: <br> | Controls: <br> | ||
1. Transform the cells with only the Tos site plasmid <br> | 1. Transform the cells with only the Tos site plasmid <br> | ||
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6. Plate on antibiotics for Tos, RFP, and N15 (plate 1) <br> | 6. Plate on antibiotics for Tos, RFP, and N15 (plate 1) <br> | ||
7. Count ratio of white colonies to red colonies <br> | 7. Count ratio of white colonies to red colonies <br> | ||
Control: <br> | Control: <br> | ||
1. Mix Tos plasmid and RFP plasmid at a 100:1 ration, transform into cells without N15 and plate (plate 2) <br> | 1. Mix Tos plasmid and RFP plasmid at a 100:1 ration, transform into cells without N15 and plate (plate 2) <br> | ||
Calculation: <br> | Calculation: <br> | ||
Enrichment = % of red in plate 1/% of red in plate 2 <br> | Enrichment = % of red in plate 1/% of red in plate 2 <br> |
Revision as of 13:56, 12 April 2010
Carolyn Kwok, Jing Luo, Dorothy Tulanont
Goals
1. Determine whether the N15 protelomerase parts are functional or not on the Tos site assuming the co-transformation is stable.
2a. If the plasmids don't stably co-transform, determine whether cells containing N15 protelomerase enrich for plasmids lacking tos.
2b. Assuming the N15 protelomerase parts are functional, determine whether the linearized "doggy bone" re-circularized in the absence of N15 protelomerase.
General Procedure
Part 1
Experiment:
1. Cotransform cells with plasmid containing the tos site and N15 protelomerase expressing plasmid.
2. Heat-shock
3. Plate with resistance plate
4. Miniprep cells
5. Digest the resulting plasmid with a restriction enzyme.
6. Analytical Mapping
Controls:
1. Transform the cells with only the Tos site plasmid
2. Run the undigested cotransformed DNA.
Part 2a
Experiment:
1. Transform cells with N15 plasmids
2. Plate on antibiotic for N15
3. Pick colonies
4. Mix Tos plasmid and RFP plasmid at a 100:1 ratio
5. Transform mixture into cells
6. Plate on antibiotics for Tos, RFP, and N15 (plate 1)
7. Count ratio of white colonies to red colonies
Control:
1. Mix Tos plasmid and RFP plasmid at a 100:1 ration, transform into cells without N15 and plate (plate 2)
Calculation:
Enrichment = % of red in plate 1/% of red in plate 2
Part 2b
4/12/10 Perform quikchange procedure on tos plasmid to correct the point mutation.