View - ResearchGate

Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 279(option 1) or bulk yeast plasmid recovery (option 2). The first protocol is generally1–3 d faster, but does not result in bankable plasmids.3.3.6.1. PCR APPROACH: RAPID SCREEN FOR INTERACTION TRAP POSITIVESA major strength of the protocol described next is that it will identify redundantclones before plasmid isolation and bacterial transformation, which insome cases greatly reduces the amount of required work. Accurate recordsshould be maintained of how many of each class of cDNA are obtained; and ifany ambiguity is present about whether a particular cDNA is part of a set or isunique, investigators should err on the side of caution.The outlined protocol includes steps of enzymatic treatment to generate crudeyeast lysates (steps 1–3), used later as template for the PCR reaction (step 4).PCR product can be obtained directly from the yeast colonies even withoutβ-glucuronidase treatment (e.g., by introducing a 10-min 94°C step at the beginningof the PCR program). However, yeast lysates obtained in this protocol alsocan be used as a source of plasmid for electroporation into Escherichia coli,instead of the more time-consuming plasmid recovery protocol described next.1. Starting from the Glu/CM Ura − His − Trp − master plate, resuspend yeast in 25 µLof β-glucuronidase solution in a 96-well microtiter plate by using a replicator. Sealthe wells using tape, and incubate on a horizontal shaker at 37°C for 1.5–3.5 h (seeNote 26).2. Remove the tape, and add about 25 µL of glass beads to each well, and seal again.Attach the microtiter plate to a vortex with a flat top surface (e.g., using rubberbands) and mix vigorously for 5 min.3. To each well, add 100 µL of sterile distilled water. Take 0.8–2 µL as a template for eachPCR reaction. Reseal the plate with tape, and keep the remainder frozen at −70°C.4. PCR amplification:a. For PCR amplification use primers specific for the library plasmid used (seeSubheading 2.7.). Perform PCR amplification (in ~30 µL volume) as follows(see Notes 27):i. 2 min at 94°C.ii. 45 s at 94°C.iii. 45 s at 56°C 31 cycles of steps ii–iv.iv. 45 s at 72°C.b. Simultaneously, perform PCR reactions from the following control templates:Empty library plasmid (diluted to about 0.1 ng); yeast from the positive controlcolonies (see Subheading 3.3.3., step 7), treated along with experimentalclones as above (see Note 28); and the same amounts of diluted library plasmidmixed together with the positive control yeast. For analysis of possibleoutcomes, see Table 3.5. Take 10 µL of the PCR product for the HaeIII digestion (below), and run out theremainder of the PCR reaction (about 20 µL) on a 0.7% agarose gel. Identify