The determination of phage titrity

in the case of excessive host cells, the number of plaques increases linearly
the number of
of phages. For this reason, the phage should be diluted before infection, not the host cells. Paving with a low MOI (infection repetition) also ensures that each phage contains only one
DNA
sequence.materials and
reagents
1. Microwave
2. LB/IPTG/Xgal
Culture
Board
3. LB
culture
4. Top layer
agar
glue gel operating steps
1. Inoculate a single ER2537 clone in a medium of 5 to 10 ml LB, shake and incubate until the middle of the growth of the number of distrogens (OD600 to 0.5)
2. When the cells grow, microwave melts the top layer of agarose gel, divided into a sterile culture tube, each tube 3 ml. Keep it at 45 degrees C.
3. Warm-up LB/IPTG/Xgal culture plate, spare.
4. Dilute the phage by 10 times the LB media.
recommended dilution range: culture of amplified phages, 108-1011; Each dilution is replaced with a new sample head, preferably aerosol blocking the gun head to avoid cross contamination.
5. Once the ER2537 culture is long until the middle of the development of the number of pairs, it is divided into trace
centrifugal tubes
, 200 ml per tube.
6. In addition to a micro centrifuge tube containing bacterial cultures to a diluted phage, only 10 ml of dilution is added to a micro centrifuge tube, rapid vortex, and 1-5min is incubated at room temperature.
7. Transfer to a tube containing a 45c top agarose gel, quickly vortex well together, immediately pour to the preheat LB/IPTG/Xgal plate, gently shake the plate to distribute the top layer glue evenly.
8. Cooling plate 5min, 37C inverted culture overnight.
9. The phage count is based on a flat dish with a number of phage spots of about 100, and the number of phages multiplied by dilution results in a titular-phage formation unit (pfu) unit (pfu) per 10 ml
phage