|
DNA Fragment Purification from Agarose or Acrylamide
For fragments from 200 bp to 10 kb the agarose purification is ideal. For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred.
Solutions
Crush and Soak Solution
500 mM NH4OAc 3.3 g NH4OAc
0.1% SDS 0.1 g SDS
0.1 mM EDTA 20 ml 500 mM EDTA
up to 100 ml with Q
store at room temperature
3 M NaOAc pH 5.2
24.6 g anhydrous sodium acetate
pH to 5.2 with acetic acid and bring up to 100 ml with Q
store at room temperature
Other Reagents
DMCS treated glass wool (Alltech Assoc. Inc. #4037, 50 g)
0.22 mm disposable micro tip filters (syringe type)
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 mg/ml) and cut out the desired band.
• 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.
| 
