result in a slightly smeared appearance to the bands produced after restriction enzyme digestion quantities of lambda phage are prepared by adding 1 3 u00d7 10 8 phage to 6 7 ml of a saturated culture of e coli strain bhb2600 grown in nzy medium and incubating at 37 u00b0 for 20 minutes the infected culture is then transferred to 330ml of nzym broth in a 2 liter flask and vigorously shaken at 37 u00b0 for about 6 hours at which time complete lysis is apparent the lysate is adjusted to 0 5 m nacl and 0 02 m mgcl 2 and cell debris is removed by centrifugation and the phage precipitated with polyethylene glycol peg 8000 lambda dna is extracted by the method of thomas et al j mol biol 91 315 1975 drosophila dna was prepared as described in tartof et al cell 37 869 1984 plasmid or lambda dna is digested at 37 u00b0 for 60 minutes with a 4 fold excess of restriction enzyme in 20 to 50 u03bcl of universal restriction buffer urb comprising 33 mm tris acetate ph 7 9 66 mm potassium acetate 10 mm magnesium acetate 100 u03bcg ml bsa 0 5 mm dithiothreitol and 4 mm spermidine all of the restriction enzymes tested possess the same activity in urb buffer as in the manufacturer 39 s recommended buffer to insert the noti linker construct into a vector of choice 5 u03bc g of the complementary strands of the appropriate unphosphorylated synthetic oligonucleotide are annealed in 10 u03bcl of ligase buffer 20 mm tris acetate ph 7 4 7 5 mm mgcl 2 0 1 mm edta 0 5.