Plaque lifts

DNA adsorption properties were first studied using a variety of solid supports for classical analysis methods including Southern and Northern transfers, dot-blotting, colony hybridization and plaque-lifts [31,32]. Studies of the interactions between nucleic acids and nitrocellulose revealed that molecular weight, finite macromolecular conformation, ionic forces and weaker forces of attraction all play a role. DNA is retained on nitrocellulose only in... 

When a bacteriophage is used as the cloning vector, the gene library is screened as an array of plaques in a bacterial lawn. A hybridization screening method is used similar to that described for plasmid screening above in this case the replica filter is called a plaque lift. ... 

Reinforced nitrocellulose (e.g., Hybond-C Super, Duralose-UV) has a tensile strength which may be 500 times more than for ordinary nitrocellulose. It has about the same binding capacity and background level as nitrocellulose. As with ordinary nitrocellulose, alkaline transfers are not possible but one or two reprobings may be possible. Nitrocellulose membranes are versatile and are preferred for colony/plaque lifts. 

Recommendations for blocking agents 5XDHS, all applications 10 X DHS, only for colony/plaque lifts BLOTTO, primarily for colony/plaque lifts yeast tRNA, primarily for RNA blots but not recommended for colony/plaque lifts homopolymer DNA, for AT- or GC-rich target DNA. 

During the last years various nonradioactive DNA labeling and detection system have been elaborated. One of them, the "DIG - DNA Labeling and Detection System" of Boehringer Mannheim is emploid in our laboratory routinely in order to probe Southern blots and plaque lifts as well as to screen bacterial colonies for recombinant plasmids. 

Plaque lifts are performed as described by Benton Davis (1977)... 

Allow the filter to remain in contact with the A phage plaques for about 4 min, then carefuUy remove (avoid lifting the top agar) and place the filter, plaque side up, onto Whatman 3MM paper previously saturated with denaturation solution. Remove trapped air bubbles and leave for 4 min then transfer onto Whatman 3MM paper previously saturated with neutralization solution and leave for 4 min. After neutralization, place the filter on dry Whatman 3MM paper and allow it to air-dry. Repeat these steps for each KP used and for each replica filter obtained from the same KP. 

The lambda phage method is readily adapted to the screening of M13 plaques (Mason and Williams, 1985). However, best results are obtained when the lifts are directly baked, without lysis and denaturation steps (M13 is 55). It is also advantageous to use ds probes or to prepare probes complementary to the strand packaged in M13. 

Lift duplicate filters from each plate, the first for 3 min and the second for 6 min (see Note 19). Store the filters plaque side up on Whatman 3MM paper until aU lifts are completed. Be sure to position registration marks carefully for accuracy in ahgning the autoradiograms later. 

One simple way to measure the static COF is to place a block of steel on a plaque of test material. The plaque is lifted on one end creating an inclined plane that is tilted higher and higher until the block starts to move as shown in Fig. 2.13. The angle of tilt can be used to resolve the forces to calculate the static COF as defined in Eq. (2.5) ... 

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