Question: I\'ve eastimated the titer of the library you\'ll be plating to be 8.5

Question

Question: I've eastimated the titer of the library you'll be plating to be 8.5 x 108 pfu/ml. You need to dilute the phage stock I give you so that you'll end up with ~500 plawues on each plate. You're only going to use 100 ul (0.1 ml) of diluted phage stock on your plate, so take that into account when planning your phage stock so that you end up with the appropriate number of phage in the right volume to plate. Keep in mind that you can't accurately pipet any volume smaller than 2 ul (and you'll be more accurate if you use 4 ul or 5 ul), and that the maximum volume that fits in an eppendorf stock tube is 1.5 ml (so telling me that your going to make your dilution by putting 0.05 ul of phage stock in 10 liters of SM phage buffer in not an acceptable answer!) I will only give your about 10-15 ul of the initial phage stock, so don't start by saying you'll put 50 ul of phage into 950 ul of SM buffer. Write our the steps in your dilution series. Plan to make one more 1:10 dilution than you need, meaning dilute the library out to the point that you can also plate what you anticipate will be 50 pfu on a plate. You will plate 2 plates at what you anticipate will be 500 pfu/plate and two plates at what you anticipate will be 50 pfu/plate, so make sure that at the end of the dilutions you have enough phage at each concentration.

Explanation / Answer

The plaque forming units:

The given stock = 8.5 x 108 pfu/ml

Let us take 10ul of the stock = 8.5 x 106 pfu and serially dilute it as mentioned below.

D1: Dilute this into 990 ul of media = 8.5 x 106 pfu ml

D2: Take 5ul and dilute it into 995 ul of media = 4.5 x 104 pfu ml

D3: Take 100ul of it and dilute in 90 ul of media = 4.5 x 103 pfu ml

Plate 100ul of D3 to obtain nearly 4.5 x 102 pfu ml = 450 pfu/ml

D4: Take 100ul of D3 and dilute into 900 ul of media = 4.5 x 102 pfu ml

Plate 100 ul of D4 to obtain 4.5 x 101 pfu ml = 45 pfu/ml

Related Questions

Navigate

• Browse (All)
• Browse Q
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.