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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-TRP tag is present. [[Image:TAP-TRPcheck.png|400px|thumb|right| PCR primers to check candidates]] This is accomplished by using a forward primer that anneals to the very 5'-end of the TAP gene you've tried to add to your gene of interest and a reverse primer that anneals to a very 3'-end of the TRP gene that was part of the TAP tag and used for selection. There are two important caveats about today's experiment. First, 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 may still see a product from this reaction. Thus we'll have to follow up in other ways to confirm that the gene of interest has, in fact, been modified. Second, you should be aware 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) as a failed tagging transformation. So don't let a negative result today fool you and 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 run the agarose gel next time. So, 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 |