2 .1 .1 .1 . L a r g e -S c a le Pla sm id P reparations
A fresh 2ml bacterial culture grown overnight in L-broth supplem ented with 60pg/m l am picillin was used to inoculate 200ml of brain-heart infusion. C ultures were incubated overnight with vigorous aeration, at 37°C. After pelleting the bacteria at 4200rpm in a J6 centrifuge for ten m inutes, they were suspended in 10ml of solution I (50mM glucose; 25m M Tris, pH8.0; lOmM EOT A) at room temperature. 20ml o f solution II (0.2M NaOH,
1% S DS) was added, followed by vigorous mixing. After 5 m inutes at 4^C, 15ml of cold solution III (5M potassium acetate, pH4.8) was added, follow ed by m ixing. After 10
m inutes at 4°C , the bulk of chrom osom al DNA and bacterial were pelleted by
centrifugation as above. The supernatant was further cleared by running it through cheesecloth, and 0.6 volum es of isopropanol added to it. Nucleic acids were allowed to precipitate for 5 m inutes on ice, and pelleted by centrifugation, as above. The pellets were dissolved in 5ml TE(10:1) pH8.0, and 5ml of 5M LiCl added to precipitate the bulk of contam inating bacterial RNA. Contents of the tubes were mixed, and let stand on ice for 5 m inutes, after which the centrifugation was repeated and supernatants recovered. Next, 25m l o f ethanol (>99.7% ) was added, tubes mixed, and incubated on ice for 5 minutes. A fter another centrifugation, the pellets were redissolved in 2.5ml TE(10:1) pH8.0. Into exactly 2.7m l of the DN A solution 4.2g of cesium chloride was added and dissolved. Next, 0.2m l o f the intercalating dye ethidium bromide [stock lOmg/ml in TE(10:1) pH7.5] w as added. The D N A -C sC l-E tB r solution was layered beneath 8ml of 55% CsCl in TE (10:1) (pH 7.5), in Q uick-Seal polyallom er tubes (Beckm an). The filled tubes were balanced within 30mg, sealed and centrifuged for at least 16 hours at 5(XX)0rpm in a fixed- angle ultracentrifuge rotor. This allows separation of supercoiled plasm id DNA from the rem aining contam inating chrom osom al DNA and RNA species. The DNA was recovered from the gradient using a needle and a syringe. The ethidium brom ide was carefully rem oved by multiple extractions with isobutyl alcohol saturated with IM NaCl. Finally the DNA was ethanol precipitated twice, the final pellet dissolved in an appropriate volume of TE(10;1) (pH7.4), and concentrations of the plasmid preparations determined.
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Selected colonies were used to inoculate 2ml brain-heart infusion containing 60^g/ml ampicillin, and these were incubated overnight in a shaker at 37°C. Bacteria were harvested by centrifugation and treated with the solutions I (lOOpl), II (200pl) and III (150pl), as in section 2.1.1.1. After this, 400pl equilibrated phenol was added to extract the proteins away from DNAs (see below), and extracted supernatants precipitated with ethanol (see below). Precipitated DNA pellets were dissolved in TE(10:1) (pH7.4) supplemented with 40pg/ml RNAase A.
2.1.1.3. Synthesis of Oligonucleotides
All oligonucleotides used in this study were provided by K.Hobbs and I.Goldsmith of the ICRF Oligonucleotide Service.
The following ohgonucleotides were used in DNA-protein interaction assays, as well as in experiments involving intracellular sequestering of specific DNA binding proteins. They were all synthesized so that following annealing, they would have a single stranded 5' GATC overhang at either end.
IFN-6 -108/-95 (NRD E) upper: GATCCAAAATGTAAATGACA IFN-6 -108/-95 (NRD H) lower: GATCTGTCATTrACATTTTG IFN-6 -104/-91 upper: GATCCTGTAAATGACATAG
IFN-6 -104/-91 lower: GATCCTATGTCATTTACAG
IFN-6 -55/-40 (NRD I) upper: GATCCTCTGAATAGAGAGAG IFN-6 -55/-40 (NRD I) lower: GATCCTCTCTCTATTCAGAC IFN-6 -33/-20 (TATA) upper: GATCCCTCATATAAATAGGA IFN-6 -33/-20 (TATA) lower: GATCTCCTATTTATATGAGG IFN-6 -777-64 (PRD I) upper: GATCCGAGAAGTGAAAGTGA IFN-6 -777-64 (PRD I) lower: GATCCTCACTTTCACTTCTC IFN-6 -647-55 (PRD E) upper: GATCGGGAAATTCC IFN-6 -647-55 (PRD E) lower: GATCGGAATTTCCC
IFN-6 -917-78 (PRD IE) upper: GATCGGTViAACTGAAAGG ffN -6 -917-78 (PRD IE) lower: G A TCCCTTFCA G TITrCC octamer upper: G ATCCATGC AAATG AA
octamer lower: GATCTTCATTTGCATG
hepoct+ upper: CGAGTGCTCATGAATATGCAAATCAATTGG
hepoct+ lower: TCGACCAATTGATTTGCATATTCATGAGCACTCGAGCT hepoct- upper: CGAGTGCTCATG/iATATCAGTCGCCATTGG
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hepoct- lower: TCGACCAATGGCGACTGATATTCATGAGCACTCGAGCT collagenase API upper: GATCCGGCTGACGTCATCAAGCTA
coUagenase API lower: GATCTAGCITGATGACGTCAGCCG somatostatin CRE upper: GATCCTGACGTCAGCCAAGGATC somatostatin CRE lower: GATCGATCCTTGGCTGACGTCAG SV40 A PI upper: GATCCTTGCTGACTAATTGAG
SV40 A PI lower: GATCCTCAATTAGTCAGCAA
Oligonucleotides used in subcloning procedures were as described in the text.
2.1.1.4. Measurement of DNA Concentration
DNA concentrations were determined by absorbance assuming 50pg dsDNA/A260 unit or 33pg SSDNA/A260 unit.
2.1.1.5. Phenol Extraction of DNA
Phenol extractions were performed to remove proteins from nucleic acid solutions. Prior to use phenol was equilibrated to pH>7.6 (Sambrook et al 1989), and an antioxidant hydroxyquinoline added to the final concentration of 0.1%. Equilibrated phenol was added to the DNA samples 1:1, the contents mixed and phases separated by centrifugation. The upper aqueous phase containing DNA was transferred to a clean tube for ethanol precipitation,
2.1.1.6. Ethanol Precipitation of DNA
To concentrate sample DNAs and/or change the buffer conditions of DNA preparations ethanol precipitations were performed. The concentration of monovalent cations in DNA samples was adjusted to either 0.25M sodium acetate (pH5.2) or 2.0M ammonium acetate, and two and a half volumes of cold ethanol was added to samples. DNA samples were stored on dry ice or in -20°C to allow the precipitates to form. The precipitates were pelleted by centrifugation at 4°C and washed with 70% ethanol followed by another centrifugation. Pellets were then air-dried and dissolved in an appropriate buffer.
DNA molecules under 200bp were precipitated in the presence of lOmM MgCl2. Whenever the amount of DNA to be precipitated was less than SOOng, either glycogen (20pg) or tRNA (20pg) was added to the sample prior to the addition of ethanol.
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