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| ==Introduction== | | ==Introduction== |
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− | To begin today’s experiment, you will “pop” some trp+ yeast from your transformation plates and then amplify the relevant portion of the released genomic DNA. The primers you will use are expected to give a ~554 base pair product only if the TAP tag is fused to the 3' region of the gene you have tried to modify. [[Image:CheckingTAP.png|400px|thumb|right| PCR primers to check candidates]] This is accomplished by using a forward primer to the very C-terminal sequence of the gene you're modifying and a reverse primer that anneals to a region within the TAP gene sequence. If the TAP tag is fused elsewhere in the genome (giving rise to the trp+ phenotype) but the SAGA- or SAGA-controlled gene remains intact, then you will see no product from this reaction. Note, however, that a negative result from these reactions (i.e. no PCR product) can just as easily be explained as a failed PCR (bad primers, dead enzyme, wrong reaction conditions, etc), and might incorrectly lead you to toss out perfectly correct samples. Only the positive result is meaningful in this experiment and we will not know the result until you see the results of the agarose gel. However, we will remain optimistic and set up overnight cultures of both the candidates you are examining today. In this way you will have cells to examine next time, when you will check them for the TAP-tag by Western blot and when you will isolate total RNA from them for microarray analysis. | + | To begin today s experiment, you will |