phage display using Stratagene's Surfzap Kit

[Alex Chang]

: Alex, We have been using this system regularly and, on average, obtain 
: primary libraries in the 10E7 range.  We use either Gigapack Gold III 
: extract or Epicentre extract (the latter is a lot cheaper and has 
: comparable efficiencies). We habitually digest with Not/Spe overnight 
: since Not1 is not the most efficient enzyme in the world.  Do you have a 
: long (8+ bases) overhang on the Not1 end?  We also tend to ligate 
: overnight at 4oC. Our background seems to vary between 10E2 and 10E3.  
: Hope some of this is useful. Barry. 

Hi, Barry:

I did all that you've mentioned, plus gel purification after 
restriction digestion using Qiagen kit. (another note: our's is 
Gigapack Gold II extract, will that make any difference?)

I am wondering whether the ratio of the insert vs lambda arms is 
important. That's the only thing I can think of (contributing to the 
wrong ligation --> low package efficiency). Any thoughts?


Alex Chang
Pathology
University of British Columbia
achang at hivnet.ubc.ca