DNA Preparation - Blunt-Ended DNA Fragments
Jeff Lawrence
5' Overhangs
1. Extract and precipitate DNA. Resuspend in:
77 µl ddH2O
9 µl 10X Medium Salt Buffer
4 µl 40 mM dNTP (10 µM each)
1 unit Klenow
2. Incubate at 25° for 30 min.
3. Heat to 70° for 5 min to inactivate enzyme.
4. Extract and ethanol precipitate.
3' Overhangs
1. Extract and precipitate DNA. Resuspend in:
17 µl ddH2O
1 µl 10X Medium Salt Buffer
2 µl 20 mM dNTP (5 µM each)
1 µl T4 DNA Polymerase (@ 5000 unit/mL)
2. Incubate at 37° for 15 min.
3. Heat to 70° for 5 min to inactivate enzyme.
4. Extract and ethanol precipitate.
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu