Recovery of DNA from Low Melting Point Agarose
Low melting point agarose gels should be poured and allowed to set up in the cold room , but electrophoresed at room temperature .
The gels should be stained at 4°C and kept cold until ready to cut out the DNA bands from the gel .
Cut out the DNA bands from the gel with new razor blades .
Use a new razor blade for each band .
Place the agarose pieces into sterile capped tubes ( the size of the tubes depends on the volume of the agarose involved ) .
Add 1X TE buffer to the tubes of agarose ( as small a volume as possible , usually about 5X the volume of the gel pieces ) .
Transfer the tubes to 37°C .
Add an equal volume of buffer - saturated phenol .
The phenol should have been warmed to 37°C .
Centrifuge the tubes at room temperature to separate the phases .
Centrifuge at 10,000 rpm , 10 min in the Sorvall or 5 min in the microfuge .
Transfer the upper aqueous layers to clean tubes .
Add an equal volume of phenol : CHCl3 : Isoamyl alcohol ( 25 : 24 : 1 ) to the tubes .
Mix well and centrifuge at 10,000 rpm , 10 min in the Sorvall or 5 min in the microfuge .
Re - extract the aqueous layer 2X with CHCl3 : Isoamyl alcohol ( 24 : 1 ) in the centrifuge as before .
Add 3 M Na acetate to a final concentration of 0.3 M and precipitate the DNAs with 2X volumes of 100 % EtOH at - 20°C overnight .
Centrifuge the tubes to pellet the DNAs .
Centrifuge in the Sorvall at 10,000 rpm , 10 min , 4°C or 10 - 15 min at 4°C in the microfuge .
Wash the DNAs 1X with 70 % EtOH in the centrifuge as before and dry the DNA pellets in the vacuum desiccator briefly ( 10 - 15 min ) to remove the EtOH .
Resuspend the DNAs with a small volume of 1X TE buffer .
Heat the tubes at 65°C for 10 - 15 min .
Mix well .
