Aplicación De Filtros En Los Rectificadores

2.2.1 Restriction digests

Restriction digests were carried out in a volume of 30 jil, using the buffer supplied with the enzyme by the manufacturer (Gibco, Promega). Digests were usually carried out at 37®C for two hours. When dual digests were carried out the lower salt buffer was first used, then the higher salt enzyme and buffer were added. Alternatively, a universal buffer was used.

2.2.2 Agarose Gels

Agarose gels for the separation of DNA fragments were generally made with 1% agarose (Gibco) in TBE, and run at 70 volts. Restriction digests, or other DNA samples, were analysed by electrophoresis through agarose gels. The percentage of each gel varied depending on the size of DNA fragments that were to be analysed. The gels consisted of agarose dissolved in either IxTAE or IxTBE and run in the same buffer. The restriction digest or DNA solution was added to the gel in loading buffer. DNA fragments were visualised by staining with the fluorescent dye, ethidium bromide which fluoresces strongly under ultra-violet light. This dye was either included in the gel at 0.5fig/ml or after electrophoresis the gel was bathed in a solution containing the ethidium bromide at the same concentration. Fragment sizes were determined by running DNA markers of known molecular weight; the Ikb ladder from BRL was generally used.

2.2.3 Purification of DNA from low melting point gels

When it was necessary to purify the product of a restriction digest away from its template or contaminating proteins or buffers, the reaction was run on a gel made with low melting point agarose (Gibco). The desired band was detected on a UV transilluminator, using the minimum possible exposure time to avoid degradation of the DNA by the UV. Using a clean scalpel the slice of gel containing the required band was excised and placed in an

eppendorf tube. The gel slice was then weighed and 5X this volume of TE was added, and the tube incubated at 65®C for 5 minutes or longer, until the gel had dissolved. Tris- equilibrated phenol was prewarmed to 37^C whilst the gel was allowed to cool to 42®C. An equal volume of phenol was then added to the agarose solution and the mixture vortexed and spun at 10,000 rpm in a microfuge for 8 minutes. The phenol was then removed and replaced with a volume of phenolrchloroform (49:1 isoamyl alcohol) and the mixing and spinning repeated. A final round with solely chloroform was then used and aspirated. 0.2 volumes of lOM ammonium acetate, 2 volumes of ice cold ethanol were then added to the remaining aqueous phase, mixed and left at -20^C for a minimum of 20 minutes. The microfuge tube was then spun at 4®C for 15 minutes at 13,000 rpm in a microfuge. The resulting pellet was washed in cold 70% ethanol and 100% ethanol and finally resuspended in TE after air drying. Approximately 10-20|il of TE were sufficient.

As the yields from this protocol were not always high, I also used a Qiagen Quick-Prep DNA extraction kit (Qiagen). The manufacturers instructions were followed throughout.

2.2.4 Infilling with Klenow

The Klenow fragment of DNA polymerase I can be used for the infilling of 3' recessed termini, a requirement for a number of cloning steps. The protocol is outlined in Maniatis et al., (1982), and requires the addition of dNTPs, each at a concentration of ImM, and the Klenow enzyme to the restriction digest products and incubation for 1 hour at 37®C.

2.2.5 Sub-cloning and ligations

Fragments of interest were subcloned in pKS (Stratagene) or pGEM (Promega) vectors for subsequent amplification and to allow in vitro transcription. Vector DNA was digested with the appropriate enzymes, the digestions checked on a gel and then the DNA was purified by one of the above methods. DNA fragments to be subcloned were also isolated in this fashion. "Sticky end" ligations generally used lug of vector DNA and 1 to

3 molar equivalents of insert DNA. The ligation reaction volume was 20 |il and contained Ix ligase buffer, ImM ATP and 1 |il of T4 DNA ligase. The ligation was carried out overnight at 16®C and the products were used to transform bacteria. If the transformations were unsuccessful the ligation reaction was run on a gel to see if any ligated product had been produced.

2.2.6 Cloning PGR products

The cloning of PGR fragments was facilitated by the use of the Promega (Madison, USA) pGEM-T Vector System. This makes use of the fact that Taq polymerase adds an adenine base to the PGR product in a template independent fashion. The vector exploits this fact via the corresponding possesion of an unpaired thymine base at the 3' end. Vectoriinsert molar ratios of 1:1 were optimal for successful ligation. Standard ligations were carried out using purified or crude PGR product. An alteration was also made to the PGR protocol when products were to be cloned, see Chapter 4 Results for details.

2.2.7 The production of cells competent for transformation

A 10 ml culture of D H 5-a bacteria was grown-up overnight (37®G with shaking). 1ml of this culture was used to inoculate 50mls of LB broth, which was then incubated for 1 hour 45 mins. at 37®G with shaking. The bacteria (split into two tubes) were then spun at 2,000 rpm for 5 mins. and each aliquot resuspended in lOmls of cold 50 mM GaGl2

and left on ice for 40 mins. Bacteria were spun again (2,000 rpm, 5 mins., 4®G) and each pellet resuspended in 2mls of cold 50 mM GaGl2. Competent cells were then either

used immediately or 140 p.1 of DMSO was added and the cells returned to ice for a further 15 mins. An extra 140 }il of DMSO was then added, the cells were aliquoted, snap frozen in liquid nitrogen and stored at -80^G. All tubes and chemicals used were autoclave sterilised.

Competent cells were thawed on ice (5-10 mins.) and transformation tubes were simultaneously cooled on ice. (The cells are very fragile and are easily damaged by shearing, reducing the efficiency of the transformation, hence care should be taken.) The plasmid to be transformed was added to the transformation tubes (approximately 10 ng of circular plasmid in TE), and 50 |il of competent cells added. The tubes were then flicked to mix and left on ice for 45 mins. The cells were then heat shocked at 37®C for 45 secs, followed by chilling on ice for 2 mins. 0.5 mis of LB broth (pre-warmed) was added to each tube and these were subsequently incubated at 37®C for 1 hr. 50-100 |il was then removed and spread on selective plates and incubated at 37®C overnight. The following bacterial strains were used in this work: XL-1 blue (Promega, USA); D H 5-a (Promega, USA) .

2.2.9 Isolation of plasmid DNA - Mini-prep

A single colony was inoculated into a 3 ml L-broth culture, containing the appropriate selection, and grown overnight in a shaking incubator at 37^C. The cells were pelleted by centrifugation at 3,000 rpm for 10 minutes. The pellet was resuspended in 200 |il of solution I. This suspension was transferred to a micro-centrifuge tube, incubated at room temperature for 5 mins and 400 |il of fresh solution II added. The tube was then inverted 5-6 times as the cells were lysed. The tubes were then transferred to ice and 300 |il of ice cold solution III added, tubes were immediately inverted to mix and allowed to stand on ice for 5-10 mins. The tubes were then spun at 13,000 rpm for 5 mins. and the supernatant removed and placed in a fresh 2 ml microtube. An equal volume of phenol rchloroform (1:1) was added, the tubes vortexed and subsequently spun again (13,(X)0 rpm for 4 mins.). The aqueous phase (750 |il) was extracted to a new microtube and an equal volume of isopropanol (propan-2-ol) added. This was mixed, allowed to stand briefly at room temperature, and then spun at 13,000 rpm for 10 mins. The pellet (generally large and easily visible - probably due to salt and/or RNA) was washed in 200 |il of cold 70% ethanol and resuspended in 40-50 |il of TE. DNase free RNase A was

either added to solution I, at the resuspension step, or during subsequent manipulations of the DNA e.g. including RNase in a restriction digest. (Failure to remove contaminating RNA makes interpretation of agarose gels difficult as large amounts of RNA increase the rate of migration of DNA bands.)

2.2.10 High quality DNA from minipreps

Alternatively, and when high purity DNA was required (e.g. automated sequencing) Qiagen mini-prep columns were used. Manufacturers instructions were followed throughout.

2.2.11 Manual DNA sequencing

DNA was sequenced enzymatically, using the USB Sequenase kit ; label incorporation was by extension reactions with ^^S-ATP.

2.2.12 Preparation and running of 50cm sequencing gels

Before use, gel plates were thoroughly cleaned with soapy water, and then rinsed with distilled water to remove all grease. The notched plate was then treated with dimethyl dichloro silane solution, rinsed with distilled water to remove traces of HCl able to interfere with polymerisation, and then allowed to air dry. 0.4mm spacers were then inserted at the edges of the plate, bulldog clips were used to maintain appropriate thickness across the whole plate, and then the sides and base of the plate were taped up. For a 50cm gel, 50ml of acrylamide/urea mix were polymerised in a beaker by addition of 90 |il TEMED (NNN'N'-Tetramethyl-l,2-diaminoethane) and 90 |il of 25% ammonium persulphate. The reagents were quickly mixed by swirling, and applied to a tilted gel plate with a 50ml disposable plastic syringe down one side of the gel plates, injecting rapidly to avoid premature polymerisation and carefully to avoid air bubbles. The comb was then inserted and secured with more bulldog clips, and the gel left to polymerise for at least an hour.

Before loading samples the gel was run for 45 minutes beforehand to achieve running temperature, and the samples were loaded after heating to 80®C for 5 minutes. The gel was run for 3 hours at 45W or 50®C. After running, the plates were separated with a spatula, the gel fixed for 15 minutes in 10% methanol, 10% acetic acid, transferred to a sheet of 3MM, covered in Saran wrap and then dried in a gel drier. Bands were visualised by autoradiography.

2.2.13 Automated Sequencing

To accelerate the processing of the sequence information an ALF Automated Sequencer was used (Pharmacia, UK). This is a semi-automated machine, in that sequencing reactions are carried out by hand, according to manufacturers instructions, and the fluorescently-labelled products are then run on a sequencing gel prepared by hand. Because of the sensitivity of the fluorescent detection system used the gels are not standard. A 6% Longranger Gel was used (J.T. Baker, UK), with 1.2x TBE in the gel and 0.6x TBE in the running buffer, the gel also contained 16.8g urea per 40 ml. To polymerise the gel 20 }il of TEMED were added and 200 |il of 10% ammonium persulphate. The sequence was then read by the ALF and the sequence captured in GCG Wisconsin format, allowing the data to be used for searching databases.

2.2.14 Sequence Comparisons

Sequences were compared using the software from the University of Wisconsin Genetics Computer Group (UWGCG). The "Gap" or "line-up" programme was used to find the optimal alignment between two predicted amino acid sequences. This programme finds optimal aligment by inserting gaps to optimise the number of alignments and uses the algorithm of Needleman and Wunsch (1970).

Oligonucleotide primers of desired sequence were ordered from a memeber of Biochemistry at ICH (Paul Rutland) and arrived as 2 ml of ammoniacal solution. A simple precipitation step was then required to liberate the primers from this solution. 0.1 volumes of 5M potassium acetate (sterile) and 2.5 volumes of absolute ethanol were added. This is done in tubes capable of high speed centrifugation. The solutions were mixed by vortexing and left at -20®C overnight. The tubes were then spun at 11,000 rpm at 4®C, for 15 minutes on a Beckman RC5Bplus centrifuge. The resulting pellet was then washed with 70% ethanol, then 100% ethanol, taking care not to displace the pellet. The pellets were then alowed to air-dry before resuspension in T.E. The concentration of oligonucleotide in the solution is critical and hence an OD (optical density) is taken at a wavelength of 260nm* This is carried out in a 1ml cuvette with l|il of oligonucleotide primer added. To determine the concentration of primer the following calculation is then used:

A260 X 1000/vol. cuvette in mis x 33 / 330 x n= }imol/ml, where n= the length of the oligonucleotide. For ng/ml multiply by the Mr (which equals 330 x n).

2.2.16 Primer Concentrations

For PGR the primers are used at 200 ngs of each primer per PGR reaction. For sequencing, 0.5 pmoles is added.

2.3 S o u th ern Blot 2.3.1 Solutions

Dénaturation solution 0.5M NaOH, 1.5M NaGl

Neutralization solution 0.5M Tris-HGl, pH 7.5, 3M NaGl. Prehybridisation solution 2xSSG, 0.1% SDS, 0.1% blocking agent Hybridisation solution 5xSSG, 0.1% SDS, 50% formamide Wash solution 5xSSG, 0.1% SDS

2.3.2 Labelling of oligo probes with for Southern blot

Forty five pmoles of probe (PCR primer, Ix kinase buffer (0.5M Tris HCl pH 7.60. IM MgCl2,50mM DTT, ImM spermidine, ImM EDTA pH8.0), T4 polynucleotide kinase, and l|il of Y - P^^ labelled ATP were combined and incubated for 1 hour. Probes were separated from unincorporated ATP using a spin column. This was a 1ml syringe packed with Sphadex G-50. This was spun for 3 mins. at ~ 1750 rpm, until dry. 200 |il of water was then added and the syringe spun again as before. The probe was then made up to 200|il and added to the column, an eppendorf tube placed under the syringe and again spun under the same conditions. Two ml of the eluent was counted on a y-counter, and probes in the range of ~ 100,000 dpm pre |il. 5-10 |il of probe were then added per hybridisation.

2.3.3 Transfer of DNA from gel to Nylon filter

An agarose gel of the appropriate strength was run as normal. This was followed by 2x 15 min. incubations in dénaturation solution, followed by 2x incubation in neutralization buffer. The DNA was then electrophoresed onto the Nylon filter (Hybond) for a minimum of 1 hour. The filter was removed from the apparatus and UV cross-linked whilst still damp. Prehybridisation solution was then added and left for 1 hour whilst rocking. This solution was then replaced with the hybrdisation solution which included 50 \i\ of the labelled probe which was incubated at 34^C for 3 hours. The filter was washed for 2 times for Ihour with wash solution and then wrapped in Saran wrap (USA) and placed in a cassette with X-ray film overnight, or for longer or shorter periods, dependent upon the signal.