protocol_281.txt

Generation of DNA fragments by DNase digestion
In a 50 µL reaction volume, resuspend 8 µg DNA in 50 mM Tris·HCl (pH 7.6), 10 mM MnCl2, 100 µg mL–1 bovine serum albumin, and 0.01 SU mL–1 DNase I.
Remove 5 µL aliquots (adding to 45 µL TE buffer, pH 7.6) 0, 0.5, 1, 2, 5, 10, 15, and 30 min after addition of the digestion mixture.
Immediately transfer to a tube containing 25 µL Tris-buffered (pH 7.0) phenol.
Perform a phenol:chloroform (1:1) extraction.
Perform a chloroform extraction.
Perform a chloroform extraction once more.
Precipitate the fragmented DNA.
Wash with 70% ethanol.
Dry.
Resuspend fragmented DNA in 23 µL of Blunt-ending Mix (100 µM dNTPs, 1 × T4 DNA Pol Buffer).
Heat at 65°C for 30 min to resuspend DNA and inactivate any DNase I that was carried over.
Cool to room temperature.
Add 2.5 U Klenow fragment and 5 U T4 DNA polymerase.
Incubate the reaction at 37°C for 1 h.
The fragmented and blunt-ended virus DNA can be run on a 1% agarose gel prior to excising fragments in the 1000–4000 bp range using a standard gel extraction procedures before downstream cloning.