Natt E, Scherer G. EMBL12, a new lambda replacement vector with sites for SalI, XbaI, BamHI, SstI and EcoRI. Nucleic Acids Res. 14: 7128, 1986. PubMed: 3020507
Shipped: ?Freeze dried bacteria-free phage lysate.
Titering:
1. Make fresh plating bacteria.? Grow E. coli host strain overnight or at least to A600 = 0.4 in medium containing 0.2% maltose (to give higher titers).
2. Spin down cells in a low speed centrifuge.? Resuspend in 0.4 volumes 10 mM MgSO4 or SM buffer.? Store at 40C.
3. Dilute phage in 10 mM MgSO4 ?of SM buffer. Mix gently because vigorous mixing reduces the titer.
4. Add 100 ul phage dilution to 100 ul prepared plating bacteria and mix gently.? Incubate in a 370C water bath for 20 minutes to allow phage to adsorb.
5. Add 3 ml LB lambda top agar (see below) containing 0.2% maltose and mix gently.? Pour onto plates.? Incubate overnight at 370C.? Fresh plates give larger plaques.
Storage:
Libraries can be frozen in liquid nitrogen with no significant loss in titer, even after repeated freeze-thaw cycles.? The libraries on dry ice can be stored at 40C or can be kept frozen at -70 0C or in liquid nitrogen.? Always freeze by plunging into liquid nitrogen.
LB Lambda top agar medium:
NaCl????????????????????????? 5 g
Tryptone???????????????? ?10 g
Yeast extract???????????? 5 g
Distilled water to??????? 1 L
Sterilize at 1210C, 15 minutes.? Cool to approximately 500C and add the following sterile solutions.
1M CaCl2?????????????????? 5 ml
MgSO4 H2O??????????????? to a final concentration of 0.2% w/v
50% maltose????????????? 5 ml
For solid? media, add 7 g. agar or agarose (for top agar) per liter or 15 g agar for basal medium prior to autoclaving.
Reference:
Biotechniques 5: 724-728, 1987.
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