further investigation.
2.3.12 A g a r o s e g e l e le c tr o p h o r e s is
PCR products were checked for specific amplification of a single product of
the correct size on 2% agarose (International Biotechnologies Inc.) gels
w ith 500 ng/ml ethidium bromide using 0.5 x TAE buffer, with Ikb ladder
(Life Technologies) as size markers. Gels were photographed on a UV
transilluminator.
2.3.13 E le c tr o p h o r e s is o f R N A in a g a r o s e - f o r m a ld e h y d e g e ls
1 pg of mRNA was electrophoresed through a denaturing agarose-
formaldehyde gel. For a 100ml gel, Ig of agarose was m elted in DEPC
98
formaldehyde to 2.1 M from a 12.33 M stock. RNA sam ples were prepared
in 1 X MOPS, 2.1 M formaldehyde, 50% form amide and 1 x RNA loading
buffer. The mixture was heated to 65°C for ten m inutes then cooled on ice
for fifteen m inutes before being loaded into the wells. The gel was run in 1
X MOPS at 60-70V for 4-6 hours.
2.3.14 N o r th e r n b lo t
After electrophoresis, the agarose-formaldehyde gel was soaked in DEPC
treated 50 mM NaOH, 0.1 M NaCl then in RNase free 0.1 M TRIS.Cl (pH 7.6) (sigma)) and finaUy in DEPC treated 2 x SSPE in each case for 20
m inutes. The RNA was transferred to hybond N (Amersham) and blotted
in 20 X SSC overnight. The lanes were marked and the filter was briefly
rinsed in 6 x SSPE. Excess water was removed from the filter which was
then exposed to 1.2 J of UV hght in a UV Stratahnker (Stratagene). The filter was then ready for hybridisation.
2.3.15 R a d i o la b e l li n g o f p r o b e s
Prohes (listed in table 2.2) were radiolabelled by the random priming method (Feinberg and Vogelstein, 1983; Feinberg and Vogelstein, 1984) as
follows: 30 to 50 ng of double stranded DNA in a volume of 30 pi was heat
denatured at 98°C for 5 minutes, mixed with 10 pi OLB, 2 pi BSA
(lOmg/ml), 50 pCi P^P]-dCTP (ICN Flow) and 2 U Klenow DNA
polymerase (NBL), and incubated at room temperature for 3 hours.
syringe plugged with polymer wool and filled with Sephadex G50. The
column was packed by centrifugation for 3 minutes at SOOg and washed
with 200 ml 2 X SSC. The probe labelling reaction was made up to a final
volume of 200 ml with 2 x SSC, loaded onto the column and centrifuged as
before. The activity of the recovered probe was measured hy counting a 2 p
1 aliquot using a Bioscan QC 2000 (3 counter.
Table 2.2. Probes used in northern analysis
Gene probe Source
GAPDH pGap Dr C. Kinnon
Cp pCp Dr C. Kinnon
mb-1 pRa3 Dr M. Reth
b29 pb29 Dr S. Muller
2.8.16 P r e h y b r i d is a ti o n o f m e m b r a n e s
Membranes were wetted in 3 x SSC before being rolled into glass
hybridisation bottles (Hybaid) interleaved with mesh (Hybaid). 10 ml of
Rapidhyb (Amersham) hybridisation solution were added and the bottles
rotated in a Hybaid oven at 65®C for 20 minutes.
2 .8 .1 7 H y b r id i s a t i o n o f m e m b r a n e s
One quarter (5 ng/ml) of the probe were denatured at 98®C for 5 minutes
and added to the hybridisation solution. Probes were hybridised to the
100
2.8.18 W a s h in g o f m e m b r a n e s a f t e r h y b r i d i s a t i o n
Membranes were washed twice in 2 x SSPE at room temperature for 20
minutes and then at 65°C for 15 m inutes in 1 x SSPE/0.1% SDS.
Membranes were then wrapped in plastic film (Saranwrap).
2.8.19 A u to r a d io g r a p h y
Membranes were exposed to X-ray film (XAR-5, Kodak) at -70% with two intensifying screens (Lightening Plus, Cronex, Dupont) for between 1 and
14 days. Films were developed on a Fuji RGII film processor.
2.3.20 R e-u se o f n y lo n f ilte r s
After autoradiography, the membranes were ‘stripped’ to remove all bound
probe, allowing re-use. The membranes were washed in boiling 0.5% SDS
solution and shaken until the solution had returned to room temperature.
The membrane was then rinsed in 3 x SSPE and autoradiographed to confirm that all the radiolabelled probe had been removed.
2.3.21 Q u a n tif ic a tio n by d e n s ito m e tr y
The intensity of the signal seen on the autoradiographs was quantified by
densitometric analysis (LKB LASER densitometer). Densitometeric
readings were plotted on graph paper and the area under the curve
measured by cutting out and weighing each curve. The expression of
normalise the values obtained for each sample. The ratio for each sample
was expressed as a percentage of the normal control.
2.3.22 S e q u e n c in g u s in g the " S equ en ase” k i t v e r s io n 2,0
Sequencing of plasmid inserts was carried out using the “sequenase” kit