This protocol was developed at The Ohio State University

1.  Run the DNA digest on an agarose gel:

• Make a 1% Seaplaque (or other very high quality) low melt agarose gel using 1X TAE prepared according to Maniatis (Add EtBr about 0.5µl stock at 10µg/ml into 50ml medium).
• Load DNA markers on the end separated from the digest of interest by 1 lane.
• Load the PI's DNA in one big lane (merged with a piece of Scotch tape).
• Run the gel at low voltage (22-23v) overnight (start at late afternoon).
• The next morning, view the gel using longwave UV, and cut the band of interest.  Place the gel slice(s) into a pre-weighted 15ml tube.  Store in the dark at 4ºC if not processed immediately.

2.  Purifying the DNA Fragment: (GeneClean Purification Technique)

• Weight the cut fragment.
• Add 3x (wt/vol) of buffer NT1 and 60 to 100ul of Glassmilk to the tube with the fragment.
• Dissolve the gel at 55ºC for 10 minutes and mix vigorously every 3 minutes.
• Place 2-4 NucleoSpin cartridges in 2ml microtubes (depends on total volume of gel slices and buffer), load 750µl dissolved gel slice onto the cartridge.  Spin at max speed for 1 minute in a microcentrifuge. 
• Discard the flowthrough.  Add up to three 750µl loads through a single cartridge, spin and discard the flowthrough.  (The cartridge has a capacity of 2µg of DNA) 
• Add 750µl of buffer NT3 to the cartridge and spin at max speed for 1 minute.  Discard the flowthrough. 
• Spin the empty cartridge at max speed for 1 minute to completely remove buffer NT3.
• Elute the DNA fragment from the cartridge: Replace tube with a fresh microtube, add 50µl of elution buffer, incubating at 37º C for 5 minutes (flick the tube at least 2X in that period), spinning down, and then repeating to get any remaining DNA.
• Precipitate the DNA overnight, using 3MTris pH 7.4 or ammonium acetate as the salt.  If 3MTris is used, incubate overnight at -20º C.  If ammonium acetate is used, incubate overnight at room temperature.
• Spin down the pellet, wash once with 70% EtOH.
• Resuspend the pellet in 20-50µl of Injection Buffer (10mM Tris-HCl, pH 7.4, 0.25mM EDTA), depending on the size of the pellet.
• Dissolve DNA at 4º C overnight.
• Determine the DNA concentration by regular 0.8% agarose gel.