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Method for determination of food phage titer In the case of an excess of host cells, the number of plaques increases linearly wi

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2018/04/16 15:06
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Method for measuring food phage titer
 
In the case of an excess of host cells, the number of plaques increases linearly with increasing phage. For this reason, the phage is diluted prior to infection rather than diluting the host cells. Plasting with low MOI (infectious repeatability) also ensures that each plaque contains only one DNA sequence.
 
Materials and reagents
 
Microwave oven
 
2. LB/IPTG/Xgal culture board
 
3. lb medium
 
4. Top agarose gel
 
Steps
 
1. Inoculate a single ER2537 clone in 5~10ml LB medium, incubate and incubate until the middle of logarithmic growth (OD600~0.5)
 
2. While the cells are growing, microwave the top agarose gel and dispense into a sterile culture tube, 3 ml per tube. Maintain at 45 ° C for use.
 
3. Preheat the LB/IPTG/Xgal plate at 37 °C for use.
 
4. Dilute the phage 10 times in LB medium.
 
Recommended dilution range: for amplified phage culture supernatants, 108-1011; for unamplified screening eluates, 101-104. Each dilution is replaced with a new one, and it is best to use an aerosol barrier to avoid cross-contamination.
 
5. Once the ER2537 culture is grown to the mid-log phase, dispense into a microcentrifuge tube, 200 ml per tube.
 
6. Add the diluted phage supernatant to a microcentrifuge tube containing the bacterial culture, add only 10 ml of one dilution to a microcentrifuge tube, vortex rapidly, and incubate for 1-5 min at room temperature.
 
7. Transfer to a tube containing a 45 ° C top agarose gel, mix quickly and vortex, immediately pour onto the pre-warmed LB/IPTG/Xgal plate, and gently shake the plate to evenly distribute the top coat.
 
8. Cool the plate for 5 min and incubate overnight at 37 °C.
 
9. The plaque count is based on a plate with a plaque number of about 100. The number of plaques multiplied by the dilution yields a titer-plaque forming unit (pfu) per 10 ml of phage.

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