cDNA library...

[fibonacci]

i juz beginning to contruct my cDNA library...

but after i study the protocol (Stratagene kit), i juz wondering how to calculate the titer of my phage???

if someone know about that, can u elaborate more about this...

 

besides that, i still don't know why i should do the mass excision...

[CharonY]

The calculation of the any phage titer is pretty much the same. Essentially you want the plaque forming units (pfu) per ml.

What you have to consider is that your original phage suspension is diluted twice. First you usually make a dilution or dilution series prior co-incubation with bacterial cells, and secondly you usually do not mix 1 ml of the whole diluted solution with the cells but only a fraction thereof.

 

So for an example:

you dilute your phage suspension 10e-4

you add 1 µl (or 1/1000th of a ml) of the resulting dilution to your bacteria

and mix everything with soft agar and count 4 plaques the next day.

 

So your pfu/ml is: 4*10e4*1000=4*10e7

 

And regarding mass excision, it depends on the purpose of your library. If you want a sample pool in order to make subtractive hybridization, for instance.