Other Reagents
DMCS treated glass wool (Alltech Assoc. Inc. #4037, 50 g)
0.22
blue tips with melted tips to serve as pestle for crushing acrylamide
Procedure
agarose gels
• Prepare spin columns by cutting off the cap of a 0.5 ml eppendorf tube and forming a hole in the bottom with a hot 18 ga needle. Fill this "mini-column" with a small ball of DMCS treated glass wool and pack down with a pipet tip.
• Cut out the desired band from an agarose gel and place in a spin column inside a 1.5 ml eppendorf tube with the top cut off.
• Spin at 6,000 rpm in a microfuge for 10 minutes.
• Phenol/chloroform extract the flow through and EtOH precipitate with glycogen or tRNA and 10% v/v of 3M NaOAc pH 5.2.
• Wash and dry, resuspend in 20 microliters TE, run 10 microliters on a gel and use 1-2 microliters for a ligation.
acrylamide
• Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10
• Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar."
• Add 1 ml crush and soak solution and incubate overnight at 37° C.
• Spin in the microfuge for 10 minutes at 14,000 rpm. Remove as much liquid as possible and add another 500 microliters of crush and soak solution.
• Repeat the spin and pool the recovered supernatant.
• Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above).
• Spin as usual, wash and dry. Resuspend in 20 microliters TE.
