Gel Assay to Determine DNA Content of Phage Lysates Protocol A crude lysate gel assay can be performed to roughly quantitate the DNA in lysates. This is often a valuable time saving step to determine if the phage yield is sufficient to warrant continuing the procedure.
Growing Bacteriophage M13 in Liquid Culture Protocol Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate.
Growth and Purification of 25-100 ug Lambda Clone DNA Protocol There are essentially three parts to this protocol: 1. growth of at least 5x10e8 pfu phage to provide an inoculum growth of a larger liquid lysate that will produce about 5x10e12 pfu; 2. concentration and purification of the phage, and; 3. DNA preparation.
Liquid Phage Lysates Protocol 5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum.
Phage Plate Stock Lysates Protocol To prepare phage lysates to be used for small or large scale phage DNA preps. This method usually produces lysates with titers of 2-8x10e10 pfu/ml.
Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage {lambda} encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses.
Streaking Lambda Phages Protocol Phage are streaked onto a medium to obtain an independent isolate prior to preparing a new lysate. This is done to reduce the likelihood of working with lysates which have become contaminated, and/or have accumulated mutations.
Titering of Bacterial Viruses Protocol When an individual bacterial virus grows in a bacterial host suspended in a top agar lawn, its progeny infect and lyse the surrounding host cells. This causes the appearance of a "hole" or plaque in the otherwise homogeneous bacterial lawn. Since each plaque represents a single virus, the number of viruses in the aliquot added to the plate is equal to the number of plaques which appear.
