Constitución del GIAP

2.11.1 .Quantitation of DNA

DNA was placed in a quartz cuvette with path length of 1 cm and the absorbance at wavelengths 260nm and 280nm was read in a spectrophotom eter. An OD2 6 0 o f 1

corresponds to 50/xg/ml double stranded DNA, 40 /xg/ml RNA and 33 /xg/ml single stranded DNA.

The ratio OD2 6 0 / 2 8 0 provides an estimate of the purity of DNA/ RNA solutions and usually

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2.11.2.Ethanol precipitation of DNA

DNA solutions were mixed with 0.1 volumes 3M Na-acetate, pH 5.2 and 2.5 volumes ethanol. After 20 min at room temperature, DNA was pelleted by centrifugation at 14,000 rpm for 15 min in a m icrofuge, w ashed tw ice w ith 75% ethanol, air-dried and resuspended in TE (lOmM Tris-HCl, pH 8.0; Im M EDTA) or water.

2.11 .S.Phenol/Choroform extraction

To remove proteins from DNA or RNA preparations, DNA was mixed with 1 volume phenol/chloroform/isoamyl alcohol (25:24:1), vortexed and centrifuged at 14,000rpm for 5min at room temperature in a microfuge. The aqueous DNA solution was re-extracted as above and then with 1 volume chloroform . The DNA or RNA solution was then precipitated with ethanol and resuspended in dHjO or TE.

2.11.4.Preparation of plasmid DNA and DNA fragments

Small scale preparations were done with the Qiaprep Spin M inprep kit, or alternatively were performed by staff of the Equipment Park, Cancer Research UK, 44 Lincoln’s Inn Fields, with a Qiagen BioRobot. Large scale purifications were done using the Qiagen Plasmid Maxi Kit. If DNA was to be used for transfections of mammalian cells, DNA was precipitated with 0.5 volumes of a PEG 6000 solution (40% (w/v) PEG 6000 in 30 mM MgClj, sterile filtered). The solution was mixed and incubated at room temperature for 10 min. DNA was collected by centrifugation in an Eppendorf centrifuge at 14,000 rpm for 20 min. The pellet was washed with 70% (v/v) ethanol and resuspended in dH2 0.

DNA fragments from agarose gels, from PCR reactions, or after endonuclease digest were purified using the Qiaquick gel extraction kit.

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2.11.5.DNA agarose gel electrophoresis

Agarose gels were prepared by dissolving 0.8-1.2% agarose in TAE buffer (40 mM Tris, 20 ml acetic acid, pH 8 , Im M EDTA). Ethidium brom ide was added to a final

concentration of 0.5 jUg/ml. DNA samples were mixed with loading buffer (60% (w/v) sucrose, 0.1 % brom phenolblue (w/v), 0.1% xylencyanol FF (w/v) in TAE) and electrophoresed at 5-20V/cm in TAE buffer.

2.11 .e.Restrictlon digest

0.2-2 fig DNA was incubated in the appropriate buffer with 10U//xg restriction endonuclease in 30 /xl for 1-2 h. PCR fragments were incubated overnight.

2.11.7.Ligation of DNA

Digested vector and insert were digested, analysed on agarose-gels and gel extracted. Ligations were performed using 200-300 ng total DNA and 200 U T4 DNA ligase (New England Biolabs) in a total volume of 10 /xl for 0.5-4 h. For blunt end ligation the BioLine Quick stick ligation buffer was used. Sometimes the ligation reaction was ethanol precipitated before transformation into bacteria.

2.11 .S.Bacterial transformation

Competent bacteria (see 2.2.1) were transform ed by heat shock. 50 /xl of competent bacteria were mixed with a ligation reaction, 50 ng of supercoiled plasmid DNA, or 1-10 /xl of a mutagenesis reaction for 30 min on ice. Bacteria were incubated for 40 sec at 42°C and incubated on ice for 2 min. 950 ml of SOC medium was added and bacteria were incubated for 1 h at 37°C. If the transformation was performed with supercoiled DNA, 10-20% of the reaction was plated onto agar plates with the appropriate antibiotics. If transformation was done with ligation or mutagenesis reactions 10 and 90% of the reaction were plated. Plates were incubated over night at 37°C.

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2.11.9.Polymerase chain reaction (PCR)

Primers were usually 18-24 nucleotides in length, plus any added sequences (restriction sites, Kozak sequences), and contained 2 GC-base pairs at the 3 ’-end. If restriction sites were added, base pairs according to the recommendations in the NEB catalogue were added at the 5 ’-end to allow for efficient endonuclease digest. Normally annealing was perform ed at 55°C. Reactions were carried out in the appropriate buffer in 50 /xl reactions. 1-50 ng of template DNA, 2/xM primers (each), 250 /xM dNTPs (Pharmacia), and 10 U Pfu polymerase (Stratagene) in 10% (v/v) DMSO were used in a MWG thermal cycler. PCR conditions were as follows:

segment 1 1 cycle dénaturation: 94°C, 2 min segment 2 30 cycles dénaturation: 92°C, 30 sec

annealing: 55°C, 30 sec extension: 6 8 °C, for 2min/kb

segment 3 extension: 6 8 °C, 5 min

2.11.10. Site-directed mutagenesis

Site-directed m utagenesis was carried out according to the Stratagene Q uikchange Manual. A pair of complementary primers was designed, 25-45 nucleotides long that resembled the sequence of the target gene with the mismatch approximately in the middle of the primer. Primers were designed with a minimum content of GC nucleotides of 40%, and so that the annealing temperature (T^) was equal to or greater than 78°C, estimated by the following formula:

=81.5-1- 0.41 (%GC) - 675/N - % mismatch

%GC: content of guanosine or cytosine nucleotides in % N: length of primer in number of nucleotides

Primers were synthesised and FPLC-purified by the O ligonucleotide D epartm ent of Cancer Research UK, Clare Hall.

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The following PCR reactions were carried out to introduce the desired mutations: 5-50 ng of template DNA were titrated in four different reactions with 125 ng of primers (each), 2.5 mM dNTPs (Pharmacia), in a final reaction of 50 fi\ with 2.5 U Pfu Turbo DNA- Polymerase (Stratagene) in the adequate buffer. The following PCR conditions were used in a MWG thermocycler.

Segment 1 1 cycle dénaturation: 95°C, 30 sec Segment 2 12-18 cycles dénaturation: 95°C, 30 sec

annealing: 55°C, 1 min

extension: 6 8 °C, 2min/kb of plasmid length

The num ber of cycles of segment 2 depended on the type of m utation that was introduced: 1 2 cycles were used for single base pair mismatches, 16 cycles for single

amino acid changes (2-3 base pair mismatches), and 18 cycles for multiple amino acid changes. 10 /tl of the PCR reactions were analysed on agarose gels, and the PCR reaction that yielded the highest amount of PCR product was digested with 10 U of D pnl (Stratagene) for 1 h at 37°C. 1-5 /xl of the m utagenesis reaction were then used to transform Epicurian Coli XL-1 Blue supercompetent cells (Stratagene). Up to 10 clones were analysed by nucleotide sequencing for the desired mutation and confirmed for their otherwise correct sequence.

2.11.11. Nucleotide sequencing

Fluorescent cycle sequencing was performed using gene specific primers (see chapter 2.4.2) and the ABI dye termination kit (Perkin Elmer). Reactions were carried out in a volume of 20 /xl containing 0.1 - 0.5 /xg DNA and 0.1 /xM prim er and 9 /xl termination mix. PCR conditions were as follows:

25 cycles dénaturation at 96°C for 10 sec annealing at 46°C for 5 sec extension at 60°C for 4 min Samples were precipitated with ethanol.

Samples were processed for electrophoresis, separated by electrophoresis and visualised by staff of the Equipment Park, Cancer Research UK, 44 Lincoln’s Inn Fields. Sequence

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analysis and alignments were done using Sequence N avigator (Perkin Elm er) and MacVector (Oxford Molecular) software.