Plaque Amplification for ELISA Samples
Selected phage clones can be identified by DNA sequencing, and target specificity can be confirmed by phage ELISA. The number of phage particles in an ELISA should be maximized. First, 20 ml phage amplifications are carried out to obtain at least 1012 pfu for each clone or pool to be used in ELISA.
- Phage Amplifications. Dilute an overnight culture of E. coli K12 ER2738 1:100 in LB to a total volume of 20 ml in a 250 ml Erlenmeyer flask, one for each clone to be tested. A clone selected at random from a titer plate of Ph.D. Phage Display Peptide Library itself can be a negative control. Alternatively, instead of analyzing individual clones, a characterization of an entire pool of selected phage can be examined for binding activity. To detect signal, it is generally necessary to amplify the third round of eluted phage, as unamplified titers are typically no more than 107 pfu/ml. To amplify a pool of phage, add 10 μl of the unamplified eluate to 20 ml of diluted overnight culture.
- Amplification of Individual Clones: pick plaques. Use a sterile wooden stick or pipette tip to stab a blue plaque from a tittering plate (note: plates should be no more than 3 days old, stored at 4°C and have < 100 plaques) and transfer to a flask containing the diluted E. coli K12 ER2738 culture. Pick well-separated plaques.
Note: instead of single plaques, add 5–10 μl of a concentrated phage to obtain more of that stock.
Follow with phage amplification cell harvest and PEG/NaCl precipitation: follow Panning Protocol 1: Solution-phase Panning with Affinity Bead Capture, Step 7.
- Resuspension. Resuspend final stocks in 50 μl. Concentrated phage stocks may be stored at 4°C for several weeks with little loss of titer. For long-term storage (up to several years), dilute 1:1 with sterile glycerol and store at -20°C. Titer the stocks for ELISA, see protocol Phage Titering. Proceed to Phage ELISA Binding Assay with Direct Target Coating.