phage display using Stratagene's Surfzap Kit
tjstbc at sd.cts.com
Tue Mar 19 11:34:53 EST 1996
We have used Gigapack II as well and haven't noticed any major drop
off in packaging efficiencies so I'm pretty sure that's not a problem.
I have recently begun setting up ligations based on pmol ends rather
than nanogram ratios and this may contribute to more efficient
ligations. Generally, I have been using 2:1-4:1 insert to arms ratio of
available pmol ends in ligation rxns. The equation for this calc. that
I have used for linear DNA is:
2(N+1) x 10E6/molecular wt. daltons where N=number of restriction
sites. 660=mol. wt. 1 base pair.
so, given that stratagene includes a test insert (1.4Kb TT binding fAB)
and suggests ligating 1uL (.25 ug) with 1.0 ug arms, this would work out
to an 8:1 ratio (ie pmol ends insert= 2(2+1)x 10E6/ 1400x660
=6x10E6/9.2x10E5 = 6.5pmol ends/ug or 1.6pmol ends/250ng. Stratagene
suggests combining this with 1 ug arms or ~0.2pmol ends for an 8:1
ratio. I have been keeping, however, to the 2:1-4:1 range with
satisfactory results. Does any of this gibberish make sense? Hope it
helps. Good luck and happy cloning. Barry.
More information about the Methods