Description
General description
Phage DNA is isolated from infected E. coli, passed through a series of enzymatic steps before final phenol-chloroform extraction. This methylated lambda DNA is partially cleaved by Bcl I, Cla I, Mbo I, Mbo II, Taq I and Xba I.
Application
Lambda Phage DNA, Methylated from Escherichia coli host strain W3110 is suitable for use as a substrate for restriction enzymes. It was used for immunoprecipitation in ChIP assays.
The lambda phage has an icosahedral head and a long tail terminating in a single fiber. At both ends of the 5′ termini are complementary 12-nucleotide single strand sequences that contribute to the cohesive ends (cos region) of the DNA. The tail of the phage latches on the host outer membrane receptor and injects phage DNA into the cell. The phage converts the E. coli to a lysogenic state in which the phage functions are repressed and the phage genome may remain dormant (prophage) for a long time. This property is seen in bacteriophages that carry CII and CIII genes that are responsible for CI expression. Bacteriophages with CI mutation in the CI gene are able to maintain a lysogenic state at defined temperatures.
Infecting E. coli strain W3110 with lambda C1857 strain creates E. coli lysogen cultures. The phage is released from E. coli cell pellets by lysing with a high salt buffer, pH 8.0. The crude mixture is passed through a series of enzymatic steps, multiple cesium gradients, and phage DNA is dialyzed against 1 mM Tris-HCl, pH 8.0, and 1 mM magnesium chloride. The DNA is finally extracted by phenol-chloroform solution.
Lambda Phage DNA, Methylated from Escherichia coli host strain W3110 has been used in DNA combing to determine the conversion factor.
Biochem/physiol Actions
Unique restriction sites: Apa I,Nae I, Nar I, Nhe I, PaeR7 I, SnaB I, Xba I and Xho I. Methylated lambda is partially cleaved by Bcl I, Cla I, Mbo I, Mbo II, Taq I and Xba I.
Preparation Note
Add the appropriate amount of 1x Tris-EDTA buffer, pH 8.0 (Product No. T9285, diluted to 1x, or comparable), to the vial.
Incubate the DNA sample vial at 65 °C for 4 hrs with frequent mixing.
After reconstitution, the product should be stored at -20 °C.
Other Notes
One O.D. (A₂₆₀) is approximately 50 μg of DNA
