pfu and phage titration determination

pfu refers
unit of
formation of phages. Here's a way to determine the effectiveness of M13:
measures phage titration
the number of phage spots increases linearly with the amount of phage added only if the infection complexity MOI (multiplicity of infection) value of the phage is much lower than 1 (i.e., when the cell overdose). For this reason, it is recommended to detect the titration of phage fluid and dilute it before infection, rather than diluting infected cells with high MOI values. Low MOI values help ensure that each phage spot contains only one
DNA
sequence.
1. Inoculation ER2738 monobacterials fall in 5-10 ml LB
culture
-base,
shaker
culture to the mid-to-mid-to-the-right (OD600 to 0.5).
2. When the cells grow, the microwave oven melts the upper layer
agaride
, divided into 3 ml of the equivalent in sterilization
test tube
, each phage dilution of one tube. Save in a spare at 45 degrees C.
3. Preheat LB/IPTG/Xgal plate at 37 degrees C, with one plate backup for each phage dilution.
4. Prepare 10x series diluted phages in LB. The recommended dilution range: enlarged phage culture clear: 108-1011; Replace each dilution with a fresh nod, and it is recommended to use a belt filter nod to avoid cross contamination.
5. When the bacterial culture is in the middle of the scale, it is divided into 200 μl of equal parts in a trace
centrifugal tube
with one tube of dilution per phage.
6. Each tube is added with a phage of 10 μl of different dilution, quickly shakes and mixes, and at room temperature is 1-5 min.
7. Add the infected cells to the upper agar culture tube at a preheater of 45 degrees C, mix quickly one tube at a time, and immediately pour on the LB/IPTG/Xgal plate at 37 degrees C preheat. Spread the upper agar evenly on a properly tilted plate.
8. After the plate has cooled down by 5 min, pour over 37 degrees C and culture overnight.
9. Check the plate and count the number of spots on the plate with to 102 phage spots. This number is then multiplied by the dilution factor to obtain an empty spot formation unit (pfu) titration per 10 sl phage.