RESULTADOS GLOBALES

All plasmids obtained from external sources were transformed into competent bacteria to make large-scale preparations for further use as described below.

Bacterial strains used

The Escherichia coli strain XL-1 Blue (Stratagene) was used to propagate and amplify all plasmid DNA described, except constructs based on the pDNA3.1 neo and pCR3.1uni plasmids (Invitrogen). Transformed XL-1 colonies were grown on under the selection marker appropriate to the vector, usually 100 pg/ml ampicillin. The Escherichia co//strain TOPF10 (Invitrogen) was used to propagate constructs based on pDNA3.1 neo and pCR3.1uni, allowed ampicillin resistance in transformed colonies. These were selected on 50 pg/ml ampicillin

Preparation o f competent bacteria

Bacteria were streaked out from a frozen stock onto L-Agar plates and incubated overnight at 37°C. A single colony was picked, inoculated into 6ml of L-Broth and grown in a shaking incubator (>200 rpm) at 37°C overnight. The following morning the bacteria were sub-cultured 1:20 into 100 ml of pre-warmed L-Broth and grown at 37°C until an OD550 of 0.48 was reached. Cells were then chilled on wet ice for 5 min before recovery by centrifugation in pre-chilled corex tubes at 6 000 rpm at 4‘’G for 5 min. The cell pellet was resuspended in 0.4 volumes of Tfbl buffer and left to rest on wet ice for 5 min. Cells were centrifuged as described before, and the cell pellet was resuspended in 0.04 times the original volume in Tfb2 buffer. The cell suspension was left on wet ice for 15 min before aliquoting into freezing vials (200 pi of cells suspension per vial), and snap frozen in an dry ice ethanol bath. For long term storage, cells were stored under liquid nitrogen or -70°C freezer.

Transformation o f competent bacteria

Competent bacterial cells were left on wet ice for approximately 15 min to thaw. For transformation of supercoiled plasmid, 40 pi of cell suspension was added to 5- 20 ng of DNA dissolved in an equal volume of water. When the products of a ligation reaction were used usually half of the reaction was used but no more than 40pl in total. The tubes were placed on wet ice. After approximately 30 min, mixing gently every 10 min., the cells were heat shocked at 42°C for 90 seconds and immediately returned to ice. 160 pi of L-Broth was then added to the cell-DNA mixture and incubated at 2>1°C for 30 min to allow expression of the drug resistance gene. Transformed cells were then spread onto pre-dried L-Agar plates containing appropriate concentrations of antibiotics and incubated upside o/n at 37°C.

Small scale preparation (mini prep) o f plasmid DNA fo r restriction digest.

Before large scale preparation of plasmid DNA, transformed colonies were analysed to ensure that the correct plasmid or ligation product was present. Single colonies were picked from a fresh plate, inoculated into 5 ml of L-Broth and grown o/n at 37°C in a shaking incubator. 1.5 ml of culture was transferred to an Eppendorf tube and spun in a microcentrifuge at 15,000 rpm for 20 sec to collect the cell pellet. The L-Broth was decanted, leaving 50-100 pi behind and then vortexed briefly to resuspend the cell pellet. 300 pi of TENS buffer was added, briefly vortexed followed by the addition of 150 pi 3 M sodium acetate pH 5.2 and vortexed again. After centrifugation at top speed in a microcentrifuge for 2 min to remove the cell debris, the supernatant was removed to a fresh tube and nucleic acid precipitated by the addition of 0.9 ml ice cold absolute ethanol. This was then centrifuged as before, the supernatant removed, and the nucleic acid pellet was washed with cold 70% ethanol. After a further centrifugation step the 70% ethanol was poured off and the nucleic acid pellet air-dried before dissolving in 30 pi water. This ‘miniprep’ DNA was then analysed by restriction digest confirm that the DNA was correct before a large scale preparation was made.

Small scale preparation (mini prep) o f plasmid DNA for sequencing.

The same protocol was used for obtaining 5 ml cultures. However, to achieve a higher degree of purity for direct sequencing mini prep DNA was made using a Quiagen miniprep kit according to the manufactures instructions or alternatively the mini prep robot (Quiagen) at the service laboratories at ICRF was employed when more than 60 minipreps had to be made.

Large scale preparation of plasmid DNA

Large scale DNA preparations (Maxi prep) were made using Qiagen maxiprep kits according to the manufacturer’s instructions. The resultant DNA pellet was washed with 70% ethanol, air-dried and dissolved in 500 pi distilled water. The concentration and purity of the plasmid DNA was determined by optical density measurement before storage at -20°C.

Restriction enzyme digestion o f plasmid DNA

Restriction endonuclease digestion of plasmid DNA was typically carried with a DNA concentration of 0.1pg/pl. Per pg of DNA 1 to 3 units of restriction enzyme were used. 10x reaction buffers were supplied with the individual restriction enzymes and used at a final 1x concentration. The optimal buffer for digests involving two restriction endonucleases was determined according to the supplier’s instructions. If no optimal buffer could be used in which simultaneous digestion by both enzymes was possible, the respective digests were carried out sequentially. Plasmid DNA was digested for a minimum of 2 hr at the optimal temperature, but where star activity was expected 0.1 mg/ml of BSA was added and the incubation time limited to two hours.

Agarose gel electrophoresis of DNA

Agarose gels were prepared by dissolving agarose at 0.8 -3.0% (w/v) in 1x TBE or TAE buffer in a microwave oven. The gel was allowed to cool to 50°C just above the indicated gelling temperature in a water bath before ethidium bromide (sigma)

was added to a final concentration of 0.1 pg/ml. Then the poured gels were allowed to set at room temperature in a gel try. DNA samples were prepared by the addition of DNA sample buffer to 1/5th final volume, and loaded into the wells of the gel submerged in 1x TBE or TAE buffer. Gels were electrophoresed in 1x TBE or TAE using a horizontal gel electrophoresis apparatus (BRL) at 5-7 V/cm at room temperature until the desired range of separation of the DNA fragments was achieved. DNA was visualised using a long wave UV light box, and photographed with Polaroid film or a digitised video camera. The sizes of fragments were estimated by comparison of their mobility relative to molecular weight markers of known size (New England Biolabs). Molecular weight markers utilised were as follows:

a) Hindlll restriction endonuclease digestion of bacteriophage X DNA.

b) Haelll restriction endonuclease digestion of bacteriophage (j)X174 DNA.

c) 1 kb DNA ladder containing bands from 1-12 repeats of a 1018 bp DNA fragment.

For preparative gels from which DNA was later isolated TAE rather than TBE was used as borate ions can interfere with this process.

Isolation o f DNA fragments from TBE-agarose gels.

Unless otherwise stated, the purification of DNA fragments from TAE-agarose gels was performed using a gel extraction kit (Quiagen). After the appropriate restriction digest, reactions were run on an agarose gel and the band of interest excised from the gel under long wave UV illumination as a thin slice. This was transferred to a pre-weighed Eppendorf tube and the weight of the gel slice noted. Elution of the DNA from the agarose gel slice was carried out as described in the manufacturer's instructions.

Spectrophometric determination of nucleic acid concentration

For quantification of DNA and nucleic acid concentration, OD readings were taken at 260 nm and 280 nm of an appropriate dilution of nucleic acid stock. An OD of 55

1.0 at 260 nm corresponds to approximately 50 pg/ml DNA, 40 pg/ml RMA and 20 pg/ml single stranded oligonucleotides. Estimates of purity were obtained by the ratio of OD260/OD280, where for pure preparations, the ratios were 1.8 (DNA) and 2.0 (RNA).

Polymerase chain reaction (PCR)

PCR reactions were used to amplify sequences from plasmid DNA and RNA by RT-PCR. All oligonucleotides used for PCR reactions were synthesised as single stranded primers by the ICRF Oligonucleotide Synthesis Laboratory.

50 ng plasmid DNA were amplified in typically 50 pi reaction: 2% deionised formamide, 0.2 mM dNTP mix of dATP, dCTP, dGTP and dTTP, 1x PCR buffer (Promega), 25pM each of 5' and 3' oligonucleotides and 0.5-2.5 mM MgCL. PCR reactions were overlaid with 1-2 drops of mineral oil to prevent sample evaporation. Typical cycling conditions included a 'hot start' of 10 min at 94"C before 1 pi (5 units/pl) Taq DNA polymerase was added and cycling according to the following schedule:

Denature 1 min at 94°C

Anneal 1 min at oligonucleotide melting temperature - 5°C Extend 1 min at 72°C.

25 cycles

Incubate at 72®C for 10 min. Store at 0°C

TA cloning

When PCR products needed to be cloned into mammalian expression vectors a TA cloning kit based on pcDNAS.I was used according to the manufactures instructions (pCRS.I TA kloning kit. Invitorgen).

After transformation colonies were picked, resuspended in 100 ml of water of which a fraction was used to inoculate 5 ml cultures while the rest was boiled for 5 min to release plasmid DNA from the bacteria. Cellular debris was removed by centrifugation at 14.000 rpm in a bench top centrifuge and typically 5pl were used as a template for an analytical PCR. Sequencing primers complementary to vector 56

sequences in conjunction with the appropriate PCR primer that mapped to the original PCR product were used. The products of the reaction were examined by agarose gel electrophoresis and colonies giving rise to the expected bands were grown up overnight for plasmid DNA preparation.

DNA sequencing

DNA sequencing was performed either using the Sequenase™ Version 2.0 DNA sequencing kit (United States Biochemical) or PRISM™ ready reaction dyedeoxy™ terminator cycle sequencing kit, both as directe by the manufacturers.

DNA ligation reaction

Vector and insert plasmid DNA were digested with the appropriate restriction endonucleases and gel-purified using the Gel extraction kit as described previously. Approximately 100 ng vector DNA was incubated with insert DNA in molar ratios of 1:1, 1:3 and 1:5 in the presence of 1x 14 DNA ligase buffer (Gibco, BRL) and 1 pi (1 U/pl) 14 DNA ligase in a 10 pi reaction volume. Sticky ligations were incubated at 16°C for at least 8 hrs while blunt ended ligations were incubated at 12°C for at least 8 hrs. Ligation reactions were made up to a 40 pi volume with water before transformation into competent bacteria.

Annealing of complementary oligonucleotides and TA cloning

This method was used to prepare expression constructs encoding very small fragments of the MUC1 gene when purification of a small PCR product would have been difficult. Double stranded oligos encoding the region of interest including a start codon and stop codon were synthesised. The sequence was prepared in a way that one base-pair overhangs compatible with the pCRS.I (invitrogen) TA cloning vector would form after annealing of the oligos.

400 pmol of each oligo with 0.4 pi of 5M NaCI were mixed in a final volume of 20 pi of water.

The tube was then placed in a beaker of boiling water, the beaker was then allowed to cool to room temperature. Then 80 pi of TE were added resulting in a

final concentration of 20mM of NaCI which was compatible with most enzyme reaction.

The double stranded oligo was then treated like a PCR product and directly cloned into the TA vector following the manufactures instructions.