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308 Serebriiskii et al.resuspend the cell pellet in 1.5 mL of fresh NM_ACI medium. Grow the cells for2 h at 37°C in this media, to allow the cells to adapt to growth in minimal mediumbefore plating, providing selection for bait (C) and library (A) plasmids.3. Serially dilute approx 100 µL of the transformed cells at least to 10 –5 in NMmedium. Plate 100 µL of each of the 10 –3 –10 –5 dilutions on:a. LB_C plates (to assess the number of bait-transformed cells).b. LB_A plates (to assess the number of library-transformed cells).c. LB_AC and NM_ACI plates (to assess the number of doubly transformed cellscapable of growing on rich and minimal medium, respectively).4. In parallel, plate the remainder of the transformed cells, dividing evenly among 10NM_ACI_5AT plates. Add 10–12 sterile glass beads per plate, Thomas Scientific(Swedesboro, NJ) or Fisher no. 11-312A, and gently agitate (do not shake) platesto distribute the cells evenly on the plate. Allow to air-dry, and then invert andincubate for 24–36 h at 37°C, followed by up to 24 h at room temperature.5. Count the colonies that grow within 24 h on the titer plates from step 3 to verifythe total number of cells transformed with the prey plasmids that were plated onthe selection plate. Calculate the efficiency of double transformation, and the totalnumber of double-transformed cells plated on selective medium. If the library isnot fully represented, it may be necessary to repeat the transformation (possiblyadjusting the amount of plasmid DNA) to achieve the full coverage of the library.However, plating more than 10 6 of transformants per plate is not recommended, asthis might result in increased spurious background.6. Inspect the NM_ACI_5AT selection plates for the appearance of colonies.a. If the predicted number of viable transformants has been plated, but nocolonies appear on the NM_ACI_5AT selection plates, one can repeat thetransformation procedure, but perform the selection at lesser stringency usinglower concentrations of 3-AT. However, note that the number of backgroundcolonies increases with lower concentrations of 3-AT.b. If too many candidates appear on the NM_ACI_5AT plates, giving theimpression of nonselected bacterial growth, one can redo the selection atgreater stringencies using higher concentrations of 3-AT (as high as 40 mM).However, caution should be used, as concentrations of 3-AT more than 30 mMmay decrease the plating efficiency of positive candidates (see Note 16).7. Colonies that grow on the selective plates should be rapidly (within 24 h) processedto the next step (see Subheading 3.2.2.).3.2.2. Confirmation of Positive Interactions:Test for Secondary Reporter ActivityOne rapid method to characterize potential positive colonies is using replicatechnique (as described in Subheading 3.1.2.) to assess the level of expressionof the secondary aadA reporter present in the selection strains. True-positives,as opposed to nonspecific mutations affecting histidine auxotrophy, should alsoshow increased aadA activity, reflected in growth on media containing streptomycin.In addition, plating potential positive clones on a series of plates made