Size (kb): 43.2000007629394500
Vector: lambda EMBL12 (phage, lambda - replacement)
Construction: lambda EMBL3 pUC12
Construct size (kb): 43.20000076293945
Features: insert detection: Spi+
vector for constructing genomic libraries
vector permitting positive selection for inserts
Phage with inserts have the Spi- phenotype.
A bacteriophage lambda vector for construction and screening of genomic libraries with a cloning capacity of 8-23 kb.
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.
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.
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.
Biotechniques 5: 724-728, 1987.