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 .
