The RFLP detected in the LCT3 PCR product was analysed directly by
digestion of the PCR product as described above. The fragments produced were
visualised by either 2% agarose gel electrophoresis (section 2.3.2) or by acrylamide gel
electrophoresis. 5pl of digestion mix were added to 5]i\ of loading buffer (either that used for SSCA, section 2.6.2.1., or for agarose gels, section 2.3.2) and loaded onto a
6% acrylamide gel in 0.5x TBE (37.5:1 aciylamide: bisacrylamide) prepared as
described in section 2.7.2.2. The gel was electrophoresed at room temperature for 30-
45 minutes at 400V, ~ 45mA. The bands were detected by silver staining (see section
The following sections describe the protocol used to detect RFLPs by the
conventional Southern blotting technique. This technique was also used to confirm the
identity of the PCR products.
2.6.1.1. Preparation o f filters.
Digested genomic DNA or PCR products, which were digested in some cases
and not others, were electrophoresed in an agarose gel as described previously (section
2.3.2.). The agarose gel to be blotted was washed twice for 30 minutes in denaturing
solution (1.5 M NaCl, 0.5 M NaOH) and twice in neutralising solution (0.5 M Tris, 1.5
M NaCl, 0.001 M EDTA, pH 7.2). A filter of Hybond N (Amersham) was laid on top
of the gel which was itself on a wick of 3MM paper soaked in 20 x SSC, a stack of
absorbent paper was placed on top of the filter and a glass plate was used as a weight.
The gel was left to capillary blot for between 3 hours and overnight. Filters were baked
at 80°C for 2 hours to fix the DNA onto the filter.
Filters of genomic DNA from the CEPH families digested with M spl were
obtained ready prepared on Hybond N+ filters from EUROGEM (the European genome
mapping initiative).
2.6.1.2. Preparation and ^ P labelling of probe DNA.
PCR products from LCT3 amplification of a genomic DNA or of pHlac5
(lactase cDNA clone) and of LACZ amplification of the plasmid pYNH24 (which was
obtained from the UK Human Genome Mapping Project DNA probe bank)(Nakamura
eta l., 1987) were gel purified by spinning through glass wool as described in section 2.3.2.1.2.. The insert from the plasmid pLBH, prepared as described above, was gel
purified by electrophoresis in low melting point agarose (Bethesda Research Labs,
Probe DNA was labelled using the Multiprime kit (Amersham) following the
manufacturers instructions. Bnelly, 20-50 ng of DNA to be labelled was boiled for 5
minutes to denature the DNA (and melt the low melting point agarose, where used) and
snap cooled on ice. The DNA was then mixed with 5 |il primers mix and 10 |il
nucleotide and buffer mix. The total volume was made up to 44 |iL 4 }il of dCTP
was then added and 2 pi Klenow polymerase. The reaction was left to proceed at room
temperature for between 5 hours and overnight. The unincorporated nucleotide was
removed from the mix by using a 'spun' column (Sephadex G-50 (Pharmacia) in TE,
prepared in a 1 ml syringe, centrifuged at 1500 g for 3 minutes). The labelled probe
was used if the incorporation was Judged to be above 50%.
2.6.1.3. Hybridisation and washing down o f filters.
EUROGEM filters and other filters were all treated in the same way. Filters
were prehybridised in 6 x SSC, 0.5 % SDS, and 5 x Denhardts solution at 65°C for
between 1 hour and overnight (100 x Denhardts comprised 2% weight to volume(w/v)
Bovine Serum Albumin (ICN), 2% w/v Ficoll 400 (PhaiTnacia) and 2% w/v
polyvinylpyrolidone). The probe and heiring sperm DNA (added to a final
concentration of 0.02 mg/ml) were denatured for 5 minutes by boiling and then added
directly to fresh hybridisation solution or a known volume of the prehybridisation
solution. Hybridisation in all cases took place overnight at 65°C.
The hybridisation solution was removed and either disposed of or stored for re
use within 1 week. Filters were then washed twice in 2 x SSC at 65°C for 10 minutes.
The filters were checked for bound radioactivity using a Geiger counter, if a higher
stringency of wash was required, filters were then washed in 2 x SSC, 0.1% SDS at
2.6.1.4. A u to rad io g rap h y .
Filters were drained ot'excess liquid and wrapped in cling tllm and then placed
in a cassette (Fuji) with intensifying screens (FG8, Fuji) and Super HR-G film (Fuji).
Registration marks were made either using radioactive ink or Glo juice (IBI) to allow
alignment of the filter and any bands on the resulting autoradiograph. Film was placed
on top and below the filter to allow multiple exposures and sealed in the light proof
cassette (Fuji). Autoradiography was carried out for between 10 minutes at room
temperature and a week at -70°C. The autoradiograph was developed by immersion in
Phenisol (Ilford) for up to 7 minutes, a stop solution of acidulated water and fixing in
Hypam fixer (Ilford) for 4 minutes (all solutions made up as recommended by the
manufacturers).
2.6.2. SINGLE STRAND CONFORMATION ANALYSIS (SSCA).
The original technique was modified to allow detection using silver staining.
To determine the best conditions for analysis of a given product the effects of
temperature, of proportion of cross linker, of 5% or 10% glycerol and o f the percentage of acrylamide used to make the gel were investigated.