Phage Display

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Jessy332
Jessy332's picture
Phage Display

Can anyone tell me roughly what numbers of scFv displaying particles one would expect to get after rescue of a single phage clone with M13KO7 helper phage? Having screened 100 colonies from phage tite plates (titre = 2 x 10e12) i have only found a single positive so far !

I wish to mix the precipitated phage from this single clone with an irrelavent library and be able to selectivly retrive my phage clone after selection so any guides as to ratios of phage clone: irrelavent phage library to mix?

ding
ding's picture
The number of phage particles

The number of phage particles rescued after amplification with helper phage generally depends on the colture volume and growth conditions (from a 100 mL 2XTY colture shaken o.n. at 30°C after infection I generally get 10e12 to 10e13 phage particles) and on the goodness of the helper phage preparation (it is generally recommended to prepare helper phage from a small rather than medium/large plaque) and the quantity of helper phage used for the infection (I usually stick to the rule of 20:1 as "phage particle: n° of E.coli cells" ratio).

As to the ratio of phage clone: irrelevant phage library particles I have no suggestion. I guess your aim is to confirm the suitability/specificity of the selected clone, but you may as well use ELISA to compare it with irrelevant library or unrelated phage clones (and monoclonal antibodies as positive controls). Good luck.