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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 ~500 base pair product only if the TAP tag is fused to the 3' region of the gene you have tried to modify. [[Image:Macintosh HD-Users-nkuldell-Desktop-colonyPCRprimers.png|thumb|right| PCR primers to check candidates]] This is accomplished by using a forward primer that anneals upstream of the gene you're deleting and a reverse primer that anneals to a region within the URA3 gene. 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 we run the gel next lab. | + | To begin today s experiment, you will |