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Table 3Suggested PCR Reactions and Interpretation of ResultsTemplate Possible outcomesEmpty prey plasmid a − + + + +Yeast containing − − − +/− +bait-prey controls bEmpty plasmid mixed − + − +/− +with yeast bait-preycontrolsClones 1…to n − − − +/− (+)Interpretation Bad mastermix/ Not enough Lysed yeast Too much yeast: As good as it gets;bad PCR template inhibited uneven template a small fractionsettings/faulty from yeast PCR load of clones alwaysamplifier failRecommendation Check or remake Add more Add less Adjust template Analyze othersolutions/repeat template/ template load/re-PCR clones until thecarefully improve lysis PCR from target number ispurified (weak) reachedbands280a PCR from the empty library vector yields a product of approx 130 bp for JG4-5 (using the FP1 and FP2 primers); approx 185 bp for YesTrp2(using the recommended YesTrp2 forward and reverse primers).b From Subheading 3.3.3., step 7 diploids. Approximate fragment sizes using the recommended primers are for Krit1, approx 1.6 kb; for Raf1,approx 2 kb; and for RalGDS, 0.4 kb.

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