IGEM:Caltech/2007/Protocols/Titering

{|cellspacing="5" cellpadding="10" style="background:#ffffff; width: 750px;"
 * -valign="top"
 * style="background:#ffffff"|

Titering

 * 1) Grow fresh overnight cultures of the bacteria of interest (i.e. LE392, D1210, etc.) [[Image:Phage_titer.jpg|right|frame|An example plate. Dark spots are cleared zones in a lawn of bacteria.]]
 * 2) Incubate the cultures until they reach an OD600 of roughly 0.1
 * 3) * More specifically, when the culture is swirled, cloudiness is observed
 * 4) Do NOT titer with bacterial cultures that have just been diluted from a culture past OD600 of 0.8 (complications will occur)
 * 5) Place agar/agarose plates in the 42 C oven and ready the 48 C water bath
 * 6) Prepare fresh 10 mM MgSO4 solution from the 1 M MgSO4 stock solution (100 ul MgSO4 in 10 mL sterile water works conveniently)
 * 7) In 1.5 mL eppendorf tubes, add 1 mL of the 10 mM MgSO4 solution.
 * 8) Prepare phage dilutions by adding appropriate amount of phage into MgSO4 solution (i.e. 1000-fold dilution is 1 ul phage in 1 ml MgSO4)
 * 9) After addition of phage to solution, invert tube at least 12 times for sufficient mixing
 * 10) For further dilutions, can simply dilute made phage dilutions (i.e. 1,000,000-fold can be obtained from taking 1 ul 1000-fold diluted phage and adding to another 1 mL of 10 mM MgSO4
 * 11) Aliquot 0.1 mL cell solution (i.e. LE392, D1210, etc.) into new 1.5 mL eppendorf tubes
 * 12) To begin infection, add appropriate amount of diluted phage to cell solution (10 ul usually works well for starters) and start the timer
 * 13) During this 30 minute infection period, label the plates and place back in the 42 C oven
 * 14) ~3 minutes before the end of infection, can take 3 mL aliquots of the top medium in 13 mL falcon tubes or 5 mL round-bottom tubes and microwave till complete liquification has occurred.
 * 15) Right before the end of infection, take out the plates and place on the bench with the cover on top. Have a flame close-by to prevent contamination.  Also, set the pippette to the right volume of infected cell solution and have sterile pipette non-filter tips ready
 * 16) Very quickly with pipette in hand, pippette out infected cell solution, add to top medium, invert the tube at least 2 times, and pour the mixed contents onto the corresponding plate.
 * 17) Rock the plate gently to allow the top medium to uniformly cover the plate and use the sterile tips to poke any bubbles.
 * 18) Let the plate cool on the bench for at least 5 minutes (10 minutes is usually enough) with the cover completely on the plate.
 * 19) Place the plate in the 37 C incubator.
 * }