Day one:
17:00: After inoculation with a single loop of bacteria inoculated in LB medium, shake at 37 ° C overnight (do not exceed 16 hours)
Day two:
7:30: Turn on the UV lamp and illuminate for half an hour (put the instrument into the fume hood); 8:00: Put 20mL of LB broth into a 250mL Erlenmeyer flask and dilute it at a ratio of 1:100. (ie put 200ul of bacterial liquid), put it into a 37 °C shaker, culture for about 3.5 hours; 11:00: turn on the UV lamp, irradiate for half an hour; 11:30: take 10μL of positive phage solution to 20mL In the bacterial culture (early logarithmic growth phase), put it into a shaker and shake vigorously (300 rpm) at 37 °C for 4.5 hours; 15:30: turn on the UV lamp and irradiate for half an hour. At the same time, the high-speed centrifuge was pre-warmed; 16:00: The culture in the Erlenmeyer flask was transferred to a centrifuge tube, centrifuged at 10,000 rpm for 10 min at 40 ° C, and the supernatant was transferred to another clean centrifuge tube and centrifuged again. Aspirate part of the supernatant and mix it with glycerin in a ratio of 1:1 and store at -200C. Pipette 80% supernatant into another centrifuge tube, add 1/6 volume PEG/NACL, overnight at 40 ° C; 17:00 inoculation with a monoclonal bacteria inoculated in LB medium, 37 ° C shaker Stay overnight (do not exceed 16 hours).
7:30: Turn on the UV lamp and illuminate for half an hour (put the instrument into the fume hood); 8:00: Put 20mL of LB broth into a 250mL Erlenmeyer flask and dilute it at a ratio of 1:100. (ie put 200ul of bacterial liquid), put it into a 37 °C shaker, culture for about 3.5 hours; 11:00: turn on the UV lamp, irradiate for half an hour; 11:30: take 10μL of positive phage solution to 20mL In the bacterial culture (early logarithmic growth phase), put it into a shaker and shake vigorously (300 rpm) at 37 °C for 4.5 hours; 15:30: turn on the UV lamp and irradiate for half an hour. At the same time, the high-speed centrifuge was pre-warmed; 16:00: The culture in the Erlenmeyer flask was transferred to a centrifuge tube, centrifuged at 10,000 rpm for 10 min at 40 ° C, and the supernatant was transferred to another clean centrifuge tube and centrifuged again. Aspirate part of the supernatant and mix it with glycerin in a ratio of 1:1 and store at -200C. Pipette 80% supernatant into another centrifuge tube, add 1/6 volume PEG/NACL, overnight at 40 ° C; 17:00 inoculation with a monoclonal bacteria inoculated in LB medium, 37 ° C shaker Stay overnight (do not exceed 16 hours).
Day three:
7:30: Turn on the UV lamp for half an hour (put the instrument into the fume hood); 8:00: Dilute the overnight culture bacteria in a ratio of 1:100 (put 50 μl of the bacterial culture into 5 ml of LB) Put in a 370C shaker and incubate for about 3.5 hours; 8:30: turn on the UV lamp, illuminate for half an hour (put the instrument into the fume hood), and let the high speed centrifuge preheat; 9:00:40 °C 10,000 rpm Centrifuge for 15 min and discard the supernatant. Resuspend the pellet by adding 1ml TBS, transfer to a microcentrifuge tube, add 1/6 volume of PEG/NACL to precipitate again, incubate on ice for 15-60min, discard the supernatant, and add 200ul TBS to resuspend the pellet, which is positive phage expansion. Add liquid. After several rounds of amplification to the required titer; 10:30: Place the IPTG/Xgal plate in a 37 ° C incubator for pre-warming, prepare a water bath and other titer supplies; 11:00: use LB medium The phage were serially diluted at 100/1000 fold. Dissolve the top coat in a microwave oven, dispense 3 mL per 3 mL in a sterile culture tube, one for each dilution of the phage, and place the tubes in a 45 ° C water bath for use; 11:30: dispense every 200 ul of bacterial culture Into the microcentrifuge tube. Each phage dilution of 10 uL was separately added to the above bacterial culture, and incubated at room temperature for 1-5 minutes. Transfer to the above sterile culture tube containing the top coat, quickly suspend, immediately spread onto the pre-warmed LB/IPTG/Xgal plate, and tilt the plate to spread the top coat. The plate was chilled for 5 min at room temperature, inverted, and incubated overnight at 37 °C.
Day four:
Plaque count, the optimal dilution of about 100 per plate. Phage titer = number of plaques × dilution (pfu/10uL)
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