Purification of DNA by High Speed Centrifugation of Agarose Gel Slices
REQUIREMENTS: T.E buffer , ddH2O,DNA Trans-illuminator, High Speed Centrifugation, Long wavelength U.V light.
PRINCIPLE: High speed centrifugation: With high speed centrifugation, the agarose matrix is compressed, and/or
...
partially destroyed by the strong force of centrifugation. The DNA molecules contained in the matrix are released in to the supernatant fluid.Function of TE buffer or dd.H2O: It is added to the gel pieces to help elute the DNA and to simultaneously wash EtBr from the DNA during high-speed centrifugation.
PROCEDURE:
1. The amplified DNA mixture was loaded in to a 1 to 1.4% agarose gel, depending on the size of the DNA, and carry out electrophoresis.
2. After electrophoresis is complete, the DNA band of interest was quickly located by illuminating the gel on a DNA trans-illluminator. The band of interest was quickly sliced out using sharp, clean razor blade.
(NOTE: avoid potential damage of the DNA molecule, the UV light should be used as briefly as possible.)
3. To enhance the yield of DNA,extra agarose gel was trimed away out side the band.(The gel sliced into tiny pieces with razor blade.)
4. The fine slices were transfered into a 1.5ml microcentrifuge tube.
(NOTE: (1) if one does not need to elute DNA out of the gel slices, the slices do not needed to be further sliced in the tiny pieces. They can be directly place in to a tube. (2) at this point, there are two options for eluting DNA from agarose gel pieces. The first option is to immediately carry out high speed centrifugation (step-5). The second option is to elute the DNA at as high a yield as possible (see below)
5. The above was centrifuged at 12,000 to 14,000x g or the highest speed at RT.
6. Following centrifugation, the supernatant fluid containing DNA was carefully transfered in to a clean microcentrifuge tube. (The DNA can be used directly for the ligation, cloning, or labeling as well as restriction enzyme digestion without ethanol precipitation. Store the DNA solution at 4° C or -20°C until use.)
Tips: (1) In order to confirm that the DNA is released from the gel pieces, the tube containing the fluid should be briefly illuminated with long wave length UV light after centrifugation. An orange-red color indicates the presence of DNA in the fluid. (2)The supernatant fluid should be immediately transfered from the agarose pellet; within minutes the temporarily compressed agarose pellet may swell, absorbing the supernatant fluid.
High Yield and Clear Elution of DNA:
1. A volume of 100 to 300 µl of dd.H2O or TE buffer was added.
2. It was centrifuged at 12,000 to 14,000xg at RT for 20 min. When centrifugation was completed, the eluted DNA in the fluid was briefly visualized by illumination using a long wave length UV light.
3. Immediately the supernatant that contains the eluted DNA was transferred in to a fresh tube.
4. The recovery of DNA was increased by, extracting the agarose pellet by re suspending the pellet in 100 µl of dd.H2O or buffer, followed by the another cycle of centrifugation.
5. The DNA supernatant was pooled and precipitate the DNA with ethanol. DNA was dissolved in 10µ of dd.H2O or TE buffer. Store the DNA at 4°C or -20°C until use. Proceed to subcloning and characterization of the DNA.
RESULT: The DNA sample was purified from the gel by high speed centrifugation.
INFERENCE: The supernatant fluid should be immediately transfered from the agarose pellet; within minutes the temporarily compressed agarose pellet may swell, absorbing the supernatant fluid.
Comment feed for this post
