Pat Heslop-Harrison, Molecular Cytogenetics, University of Leicester PHH4@REMOVEle.ac.uk
A 20 µ l of reaction is convenient for electrophoresis in agarose gel, but scale can be adjusted.
- Pipette the following into a 1.5 ml clean microcentrifuge tube on ice NA in TE buffer (for BAC DNA, 5-10 µ l (ca. 200 µ g of DNA), measure the DNA content previously with spectrophotometry ) of 10 × restriction buffer (use corresponding buffer to the enzyme, e.g., for Hin dIII, use Buffer B(Roche) or NE Buffer 2 (BioLab))
H 2 O to 20 µ l (high quality, e.g., water purchased from Sigma)
- Add 0.3 µ l restriction endonuclease and incubate overnight (or at least 3 hr) at 37 ° C. DNA should be digested completely. Crude DNA preparations may require more enzyme. The volume of enzyme should not exceed 1/10 the volume of the final reaction mixture, because glycerol in the enzyme storage buffer may interfere with the reaction.
- Stop the reaction and prepare it for electrophoresis by adding 2 µ l (10% of reaction vol) of 10 × loading buffer.
- Run on agarose gel, stain with ethidium bromide and photograph. Select agarose content (see below). For Southern blotting 0.5 % agarose gel is recommended and run slowly with 5 V per cm gel length (e.g. 50V for 10 cm gel). For most applications, we prefer TBE buffer (more buffering capacity but more expensive than TAE).
Appropriate agarose concentrations for separating DNA fragments of various sizes
|Agarose (%, w/v)
||Effective range of resolution of linear DNA fragments (kb)
||30 to 1
||12 to 0.8
||10 to 0.5
||7 to 0.4
||3 to 0.2
can go up to 7% for resolution under 100 bases, but note the cost is >£5 $USD10 per gel, and acrylamide electrophoresis may be more appropriate