Flint Group Protocols
5. PHENOL-CHLOROFORM
‘CLEANING UP’ OF DNA SAMPLES
1) Transfer sample to fresh Eppindorf tube, clearly labelled. Double
the volume of the sample by adding 1:1 phenol : chloroform – e.g.
add 100μl of phenol and 100μl of chloroform to a 200μl sample of
DNA, making a total volume of 400μl.
2) Mix contents of tube by finger flicking or inverting, then spin at
11,000 RPM for 5 minutes.
3) Transfer the upper layer to a clean Eppindorf, being careful not to
disturb the interphase. Repeat steps 1) –3), if necessary.
4) Fill the tube with cold absolute (100%) ethanol, adding about 1ML
to the tube. Mix WELL by finger flicking and inverting the tube.
Leave in the freezer for at least 15 minutes and then spin at 11,000
RPM for 15 minutes. NB: If the DNA cannot be seen after mixing
with ethanol, then be sure to note how the tubes are arranged in the
centrifuge before spinning, so that you know roughly at which
position the DNA will form a pellet in each tube.
5) Remove the supernatant using a P1000 pipette and add 1 ML of
cold 70% ethanol to the pellet. Spin again at 11,000 RPM for 10
minutes.
6) Remove the supernatant as before. Air dry the tubes on tissue paper
or dry them on the hot block at 37oC (this will take less time).
7) Resuspend the pellet in 100μl of TE Buffer (I used 10mM TrisHCl,
pH 7.5 and 1mM EDTA).
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