T7Select - Anyone familiar with T7Select for library construction (Aug/02/2007 )
does anyone have any experience with making a library (specifically a random peptide library) using Novagen's T7Select system. It's been around for ages, but I don't see many people using it. Is the library quality one normally gets too low to bother with it?
I'm contemplating about using either the Ph.D. phage display libraries from New England Biolabs or making my own random peptide library using T7Select and the latter seems to have some advantages when the library is actually made. But is it really that difficult to do or do people just like to start working with a pre-made library instead of making it themselves?
Any info will be greatly appreciated.
If anyone is actually reading this stuff, I'll answer myself and maybe help someone else down the road. I did decide to make my own library. The whole process took a couple of months of work. The library, when it is made, does work and is really nice and robust to use.
I would like to point out a couple of things though:
1. the packaging efficiency is not quite as high as the gents from Novagen tell you in their publications (duh!!) but it comes pretty close. You should count on a packaging efficiency of about 1-5 x 10^7 pfu/ug when packaging ligation reactions.
2. the displayed moiety (be it peptide or protein) is displayed C-terminally whereas the peptides in NEB's libraries are displayed N-terminally. There are pluses and minuses for both. It depends on what type of interaction you looking at. C-terminal expression is a major plus when making cDNA libraries, because in N-terminal display you cannot get any STOP codons if you want functional expression.
Other aspects of the experimental set-up are more or less the same with the exception of one or two minor differences, which should not incline anyone to choose one display format over the other.