Phage amplification, titration measurement method

day
17:00: After inoculation ring to take a monoclonal bacteria inoculated in LB
culture
liquid, 37 degrees C
shaker
overnight (no more than 16 hours)
second Day
7:30: Turn on the UV light and shine for half an hour (put the device in the ventilation cabinet); 8:00: Put 20mL LB culture fluid in a 250mL triangle
sling
, press 1:100 Dilute the fine culture overnight (i.e. put in 200ul bacterial liquid) and place it in a 37c shaker for about 3.5 hours; 11:00: turn on the UV lamp and shine for half an hour; 11:30: take 10 sl positive
phage The bacteria
liquid is added to 20mL bacterial culture (early in the series), then placed in the shaker at 37 degrees C shaking violently (300 rpm) for 4.5 hours; At the same time, let the high-speed
centrifuge
pre-warm; 16:00: transfer the culture in the triangle burner to a
centrifuge tube
, 40 degrees C, 10,000 rpm centrifuge 10min, will be transferred to another clean centrifuge tube, centrifugation again.
the absorbing part on the 1:1 ratio and glycelycere mixed preservation species, placed in -200C to save. Absorb 80% of the upper clear into another centrifuge tube, add 1/6 volume PEG/NACL, overnight at 40 degrees C;
Day 3
7:30: Turn on the UV light and irradiate the device for half an hour (putting the device in the ventilation cabinet); 8:00: Dilute overnight cultured bacteria at a scale of 1:100 (put 50 ml of bacterial culture into 5 ml LB) and put it in 370C shake Bed, cultured for about 3.5 hours; 8:30: Turn on the UV lamp, shine for half an hour (put the device in the ventilation cabinet) while preheat the high-speed centrifuge; 9:00:40 degrees C 10,000rpm centrifugation 15min, discard. Add 1 ml of TBS re-hanging precipitation, and transfer to a trace centrifuge tube, add 1/6 volume of PEG/NACL precipitation again, incubate 15-60min on ice, centrifuge abandoned clear, plus 200ulTBS re-suspended precipitation, this is positive phage amplification.
expanded to the required titration in several rounds, 10:30: put the IPTG/Xgal plate into a 37c
ringer
box to preheat, prepare drip-measuring supplies such as water baths, and 11:00: dilute phages with LB culture with 100/1000x series. Dissolve the top layer of glue in the microwave oven, each 3mL is packed in a sterile culture tube, one phage per dilution, put these culture tubes in a 45 oC water bath for backup; Each phage dilution of 10uL was added to the above bacterial culture and incubated at room temperature for 1-5 minutes. Transfer to the sterile culture tube with the top layer glue above, quickly mix and suspend, immediately spread to the preheat LB/IPTG/Xgal flat dish, tilt flat dish so that the top layer of glue spread smoothly. Cool flat dish 5min at room temperature, upside down, cultured overnight at 37C.
the fourth
, the phage spot count is optimally diluted at about 100 per flat dish. Phage titration - The dilution × (pfu/10uL)