ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

of 5M NaCl (Ausubel et al., 1999) was added and the solution was mixed thoroughly.
Then 3ml of CTAB/NaCI
solution (Ausubel et al., 1999) was added and mixed. This was
incubated for Ih in a 65°C waterbath. Following incubation an equal volume (14.85m1)
of 24: 1 chloroform/isoamyl
alcohol was added. The solution was allowed to extract for a
few minutes and then centrifuged for 10 min at 6000 g (7000 rpm) at room temperature.
The supernatant was transferred to a fresh tube with a wide bore pipette (5m1 Gilson
pipette). Isopropanol (0.6 vol) was added to the supernatant and then mixed gently until
the DNA precipitated. This was centrifuged at 10,000 g (9000 rpm) for 5 min and the
supernatant discarded. The pellet was resuspended in 1 ml of 70% ethanol and washed
and then centrifuged again at 10,000 g (9,000 rpm) for 10 min and the supernatant was
discarded again. The pellet was resuspended in 3ml of TE buffer and stored at 5°C
overnight to allow the pellet to resuspend in the buffer. The following day the solution
was transferred to a glass bijou bottle and stored at 5°C to prevent degradation of DNA.
To determine if chromosomal DNA was extracted, 20µ1 of the solution was mixed with a
5x loading dye and then electrophoresed on a I% agarose gel (TAE buffer) at 80V for 30
minutes (section 2.9.2).
2.9.2 -
Preparation, electrophoresis and recording of agarose gels
This basic method (Sambrook et al., 1989) was used for agarose gel
electrophoresis throughout the project. The running voltage and length of running period
were often altered for the varying types of analysis this protocol was employed in. For
example, when assessing chromosomal DNA, the voltage was high (e. g. 100V) and as a
result the running time was short, but when analysing small DNA fragments from a
restriction digest, it was necessary to run the gel at a low voltage (20V) often overnight to
help maintain distinct bands for better analysis.
For electrophoresis, 1x TAE electrophoresis buffer was prepared from 50x stock
solution (Sambrook et al., 1989), which was used as running buffer and gel buffer. A 0.8-
1% TAE agarose gel (Hi-pure, low EEO, Bio-Gene) was prepared and pre-stained using
0.5 µg ml-1 ethidium bromide (Sigma) for DNA visualisation. Two sizes of gel casting
tray (Anachem Origo), 50ml (6 x7 cm) and 150m1(13 x 15 cm) and 12 well and 16 well
casting combs (Anachem Origo) were used. The samples and standards were prepared in
sterile 1.5m1 Eppendorf tubes by mixing a 1: 5 ratio of 5x loading buffer (Roche) to
42