In the case of excessive host cells, the number of plaques increases linearly with the increase of phage. For this reason, before infection, the phage should be diluted, not the host cells. Plating with low MOI (infection repeatability) also ensures that each plaque contains only one DNA sequence.
Materials and reagents
Microwave oven
2. LB / IPTG / Xgal culture plate
3. lb medium
4. Top agarose gel
Steps
1. Inoculate a single ER2537 clone in 5 ~ 10ml LB medium and incubate with shaking until mid-logarithmic growth (OD600 ~ 0.5)
2. As the cells grow, microwave the top agarose gel and dispense into sterile culture tubes, 3ml per tube. Maintain at 45 ℃ for use.
3. Preheat the LB / IPTG / Xgal culture plate at 37 ° C and set aside.
4. Dilute the phage 10 times in LB medium.
Recommended dilution range: culture supernatant of amplified phage, 108-1011; screening eluent for unamplified, 101-104. Use a new tip for each dilution. It is best to use an aerosol barrier to avoid cross-contamination.
5. Once the ER2537 culture grows to the middle of the logarithmic growth phase, aliquot it into microcentrifuge tubes, 200ml per tube.
6. Add the diluted phage supernatant to the microcentrifuge tube containing the bacterial culture. Add only 10ml of one diluent to a microcentrifuge tube, vortex quickly, and incubate at room temperature for 1-5min.
7. Transfer to a tube containing 45 ℃ top agarose gel, vortex and mix quickly, immediately pour onto the preheated LB / IPTG / Xgal plate, and gently shake the plate to evenly distribute the top layer gel.
8. Cool the plate for 5 min and incubate at 37 ° C overnight.
9. The plaque count is based on a dish with a plaque count of about 100. Multiply the plaque count by the dilution to obtain the titer-plaque forming unit (pfu) per 10ml of phage.
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