3 Purification of Nucleic Acids From Eukaryotic Cells
59
2b) Cells growing in suspension: Recover the cells by centrifugation at 1500
g for 10 min, 4°C. Resuspend the cells in a volume of ice-cold TBS equal
to the volume of the original culture. Recover the cells by centrifugation, repeat the washing and proceed as described above.
2c) Tissue samples: Excise and immediately mince tissue quickly and freeze
in liquid nitrogen. Grind 200mg to 1g tissue with prechilled mortar and
pestle, or crush with hammer to fine powder. Add little by little 1 Ovol of
extraction buffer in a beaker. Allow powder to spread over the surface of
the extraction buffer, and then shake the beaker to submerge the material. When all of the material is in solution, transfer it to a SOml centrifuge tube, incubate for 1h at 37°C, and then proceed to step 3.
2d) Blood samples: Collect approximetaly20ml offresh bloodin tubes, containing 3.5 ml of ACD. The blood may be stored at 0°C for several days
or at -70°C indefinitely before the DNA is prepared.
- Fresh blood (20 ml): Spin at 1300 g for 15 min and discard supernataut plasma. Carefully transfer the buffy coat (a broad band of
white blood cells ofheterogenous density) to a fresh tube and recentrifuge. Resuspend the buffy coat in 15 ml of extraction buffer. Incubate for 1h at 37°C, and proceed to step 3.
- Prozen blood (20 ml): Thaw in a water bath at room temperature,
transfer 10 ml to a 50 ml centrifuge tube, and mix with 2 vol of
PBS. Then spin the blood at 3500 g for 15 min at room temperature
and discard the supernatant, which contains lysed red cells. Resuspend pellet in 15 ml of extraction buffer, incubate for 1h at 37"C, and
proceed to step 3.
3. Add proteinase K to a final concentration of 100 ).tg/ml and incubate at
sooc for 3h or at 42°C overnight in a water bath. Pipette up and down the
solution periodically with a wide-bore pipet 5 times.
Note: Only if sample is transparent, proceed to step 4. Otherwise, the granular cell fragmentswill trap most of the DNA during the subsequent extraction and centrifugation.
Note: Incomplete removal of all traces of proteinase K by subsequent phenol
extraction can result in DNA that is refractory to digestion with resctriction
endonucleases.
4. Cool the solution to room temperature, and add an equal volume of
phenol:chloroform:isoamyl alcohol (phenol >pH 7.8) (see Subprotocol
1) and gently mix the two phases by slowly turning the tubeend over
end for 10 min or by rotating slowly (20 rpm) on a wheel.
59
2b) Cells growing in suspension: Recover the cells by centrifugation at 1500
g for 10 min, 4°C. Resuspend the cells in a volume of ice-cold TBS equal
to the volume of the original culture. Recover the cells by centrifugation, repeat the washing and proceed as described above.
2c) Tissue samples: Excise and immediately mince tissue quickly and freeze
in liquid nitrogen. Grind 200mg to 1g tissue with prechilled mortar and
pestle, or crush with hammer to fine powder. Add little by little 1 Ovol of
extraction buffer in a beaker. Allow powder to spread over the surface of
the extraction buffer, and then shake the beaker to submerge the material. When all of the material is in solution, transfer it to a SOml centrifuge tube, incubate for 1h at 37°C, and then proceed to step 3.
2d) Blood samples: Collect approximetaly20ml offresh bloodin tubes, containing 3.5 ml of ACD. The blood may be stored at 0°C for several days
or at -70°C indefinitely before the DNA is prepared.
- Fresh blood (20 ml): Spin at 1300 g for 15 min and discard supernataut plasma. Carefully transfer the buffy coat (a broad band of
white blood cells ofheterogenous density) to a fresh tube and recentrifuge. Resuspend the buffy coat in 15 ml of extraction buffer. Incubate for 1h at 37°C, and proceed to step 3.
- Prozen blood (20 ml): Thaw in a water bath at room temperature,
transfer 10 ml to a 50 ml centrifuge tube, and mix with 2 vol of
PBS. Then spin the blood at 3500 g for 15 min at room temperature
and discard the supernatant, which contains lysed red cells. Resuspend pellet in 15 ml of extraction buffer, incubate for 1h at 37"C, and
proceed to step 3.
3. Add proteinase K to a final concentration of 100 ).tg/ml and incubate at
sooc for 3h or at 42°C overnight in a water bath. Pipette up and down the
solution periodically with a wide-bore pipet 5 times.
Note: Only if sample is transparent, proceed to step 4. Otherwise, the granular cell fragmentswill trap most of the DNA during the subsequent extraction and centrifugation.
Note: Incomplete removal of all traces of proteinase K by subsequent phenol
extraction can result in DNA that is refractory to digestion with resctriction
endonucleases.
4. Cool the solution to room temperature, and add an equal volume of
phenol:chloroform:isoamyl alcohol (phenol >pH 7.8) (see Subprotocol
1) and gently mix the two phases by slowly turning the tubeend over
end for 10 min or by rotating slowly (20 rpm) on a wheel.