Phenol extractions were performed to remove contaminating protein from samples of nucleic acids. An equal volume of phenol/chloroform/iso-amyl alcohol (25:24:1)
containing 0.1% (v/v) hydroxyquinolone was added, the sample vortexed briefly, and the phases separated by centrifugation (20,00Cfc) in a microcentrifuge for 5 min. The upper
aqueous layer was removed to a fresh tube and the sample was usually back-extracted with
0.5 volumes of distilled water. This was then combined with the first extraction and the
nucleic acids precipitated with alcohol (see Section 2.8.2).
2 J J . Precipitation of DNA
All precipitations o f nucleic acids, unless otherwise stated, were carried o u t in the following manner.
DNA solutions were adjusted to 03 M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol or 1 volume of isopropanol added. Precipitations were carried o u t either
Chapter 2 53
adds were recovered by centrifugation (microcentrifuge) for 5-15 min at 20,00Qj. The pellets were washed once in 70% ethanol (0.9 ml) and dried briefly under vacuum before
resuspension in TE buffer (lOmAf Tris-HCl (pH 7.6], 1 mM EDTA) or sterile distilled water.
2 J J . Gel electrophoresis of DNA
i) . Agarose gels.
DNA was routinely analysed for purity or following restriction enzyme digestion by electrophoresis through horizontal agarose slab gels submerged in 1 x TAE buffer (40 mM
Tris-acetate, 1 mM EDTA). The percentage of agarose, made up in 0.5 x TAE, was varied between 0.8 and 2.0% (w/v) depending upon the size of the DNA fragments to be resolved.
Electrophoresis was performed using a Gibco-BRL Model H5 gel tank which allowed gels to be poured in a plastic tray of dimensions U.Ox 14.0 cm (width x length). Wells were
formed using 1 or 2 mm thick combs of 10 or 20 teeth depending upon the number or volume of samples to analysed. Samples were supplemented with one fifth volume of load dye (5 x TAE, 30% [v/v] glycerol, 0.25% [w/v] bromophenol blue) and electrophoresis performed at 50-150 V at room temperature with buffer containing O Ajig/m l ethidium bromide. Size markers were included in all gels; at least one track contained 1-4 fig of Gibco-BRL 1 Kb ladder (fragment sizes ranged from 12-0.1 Kb). DNA was visualised by
placing the gel on a UV trans-illuminator and photographed using a Polaroid camera and
type 55 4" x 5" land film.
ii) . Extraction o f DNA from agarose gels
DNA was purified from agarose gels by electro-elution (Sambrook et aJ„ 1989). A dialysis membrane was first rinsed with 0.5 x TAE, sealed at one end with a dialysis clip and
filled with this buffer. The region of the gel containing the desired DNA fragment was excised with a clean scalpel and placed into the dialysis bag. Excess fluid was removed,
Chapter 2 54
the bag sealed with another clip. The bag was then submerged in electrophoresis buffer ( l x TAE) in the gel tank and a voltage (100-200 V) applied for 1-2 hrs. The electrophoretic
removal of the DNA from the gel slice was checked by UV trans-illumination and the polarity was reversed for 30 s to free the DNA from the dialysis tubing. The buffer
containing the DNA was removed to a microcentrifuge tube and the bag rinsed out with
0.5 volumes (approximately 200 p\) of 0.5 x TAE. The eluted DNA was subsequently extracted with phenol/chloroform/iso-amyl alcohol (25:24:1) and ethanol precipitated
before use in further reactions.
H i). Polyacrylamide gels
Native polyacrylamide gels were employed in the preparation of end-labelled DNA
probes or, at low ionic strength, for the resolution of nucleo-protein complexes in gel
retardation assays. Radiolabelled RNA probes were also purified by electrophoresis through denaturing 8 Af Urea gels. In all cases 4 or 5% (w/v) gels (20:1 acrylamide:bis-acrylamide) were formed in a 150x170 x 0.8 mm (width x length x thickness) apparatus and polymerized with 200p \ (w/v) ammonium persulphate and 25 /¿l TEMED for 1 hr. Electrophoresis was carried out in a Gibco-BRL
model V16 system at 150 V in 1 x TBE (89 mAf Tris-HCl [pH 8.3], 89 mAf Boric acid,
10 mAf EDTA) or 0.2 x TBE for low ionic strength gels.
iv). Extraction o f DNA from polyacrylamide gels
Radiolabelled DNA fragments or oligonucleotides were recovered from
polyacrylamide gels by the 'crush and soak' method (Sambrook et al., 1989). The gel slice containing the DNA was placed in a microcentrifuge tube and crushed against the side with
a pipette tip. One to two volumes of elution buffer (0.5 Af ammonium acetate, 10 mAf magnesium acetate, 1 mAf EDTA [pH 8.0], 0.1% [w/v] SDS) was added and the sample
shaken overnight at 37"C on a rotary platform to elute the DNA. The sample was then
Chapter 2 55
buffer was added and the procedure repeated. Both supernatants were then combined and
contaminating acrylamide fragments removed by passage over siliconized glass wool. A
1 ml syringe was loosely packed to 2-3 cm height with glass wool and placed in a 15 ml Corex tube above a microcentrifuge tube. The sample was pipetted into the syringe body
and forced over the glass wool by centrifugation at 50Q? for 1 min. The solution was
collected and subsequently extracted with phenol/chloroform/iso-amyl alcohol (25:24:1), ethanol precipitated, washed with 70% ethanol, re-precipitated and washed with 70%
ethanol twice more in order to remove acrylamide contaminants. After each step excess ethanol was removed from the samples by brief re-centrifugation and aspiration with a
micro-pipette. Samples were then finally resuspended in TE or sterile distilled water for further use.
2.8.4. Use of DNA modification enzymes
i) . Restriction enzyme digests
The DNA to be cleaved by the restriction enzyme(s) was prepared in sterile distilled
water and the appropriate 10 x buffer concentrate added. Enzymes were supplied by either Gibco-BRL or Amersham and the specified manufacturer's buffers were used in all cases.
The number of units of enzyme required to digest the DNA in the given time (1-24 hrs) was
calculated from the activity of the enzyme with aDNA as the substrate. This was then added
to start the reaction and the tube incubated at the required temperature.
ii) . Dephosphorylation o f DNA
Following digestion with restriction endonucleases plasmid vector DNA was usually
treated with C1AP to the remove 5' terminal phosphates and prevent re-ligation. Digests were first extracted with an equal volume of phenol/chloroform/iso-amyl alcohol (25:24:1),
back extracted with 0.5 volumes of distilled water and ethanol precipitated. The pellet was
resuspended in 50/d CIAP buffer (10 mM Tris-HCl (pH 8.3], 1 mM ZnCl 3 1 mM MgCl 2) and for protruding 5' ends, 1 unit of CIAP per lOOpicomoles of DNA was added and
Chapter 2 54
incubated for 30 min at 37°C. Blunt or recessed termini were incubated with 1 unit/2 p/comoles for 15 min at 37°C, then with a second aliquot of CIAP for a further
45 min at 45°C (Sambrook et al., 1989).
iii). Blunt end reaction
DNA molecules possessing protruding 5' termini generated by restriction enzyme
digest were converted to blunt ends when required by incubation with the Klenow fragment
o f E.coli DNA polymerase I in the presence of the required nucleoside triphosphates. The restricted DNA was extracted with phenol/chloroform/iso-amyl alcohol (25:24:1), back
extracted, ethanol precipitated and resuspended in buffer (0.5 M Tris-HCl (pH 7.5], 0.1 M
MgSO,*, 1 mM DTT, 500 pg/m l bovine serum albumin {fraction V}.) containing the dNTPs at 1 mM final concentration. The reaction was started by the addition of 1 unit of enzyme per pg of DNA and incubated at room temperature for 30 min. This was then terminated with 1 /al 0.5 M EDTA (pH 8.0) followed by extraction with phenol/chloroform/iso-amyl alcohol (25:24:1), back extraction and ethanol precipitation.
/v). Ligation
The ligation of DNA fragments was performed as follows: one hundred
microgrammes of vector DNA was mixed with an equimolar amount of insert DNA, containing compatible termini, in a final volume of 50^1 ligase buffer (50 mA# Tris-HCl
(pH 7.6], 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 5% (w/v) polyethylene glycol-8000). One Weiss unit of T4 DNA ligase was added and the reaction incubated at 16°C overnight.
Cohesive and blunt-ended termini were ligated in the same way.