FUNDAMENTACION TEORICA

2.3.1 Enzyme reactions

DNA was digested with restriction endonucleases (Life Technologies; Promega, Southampton, UK), using the manufacturers’ recommended reaction buffers and temperatures. Multiple digests were carried out in buffers compatible with the different endonucleases used. Digestion volumes varied depending on the amount o f DNA digested. In general, total volumes o f 20-40 pi were composed o f 10% DNA (0.1 -5 pg), Ix buffer, 2-5 units o f enzyme/pg DNA and distilled water (dH20). A unit is defined as the amount o f restriction enzyme necessary to digest 1 pg o f DNA in 1 h.

In order to ligate incompatible restriction sites, double-stranded DNA ends were blunted by filling in with 1-5 units o f DNA Pol I Large Klenow fragment (Promega)/pg DNA, the recommended reaction buffer and 0.05 mM deoxynucleotides (dNTPs), in a total volume o f 20-60 pi, for 30 min at 37°C. The reaction was stopped at 75°C for 10 min. Alternatively, the DNA was blunted with 0.5-1 unit o f Mung Bean endonuclease (Life Technologies)/pg DNA in Ix reaction buffer for 20 min at 30°C. The reaction was stopped by addition o f gel tracking dye (next section).

To prevent self-ligation o f vectors linearised with a single restriction enzyme, 5 ’ phosphates were removed fi-om the DNA fragments by using calf intestinal alkaline phosphatase (CIP; Life Technologies). The linearised plasmids were incubated with 1 unit CIP/pg DNA, in a total volume o f 20-50 pi, for 30 min at 37°C. The reaction was stopped at 75°C for 10 min.

Enzymes were removed with the QIAquick PCR Purification kit (QIAgen, Crawley, UK), according to manufacturer’s instructions. The protocol ensures removal o f primers <10 bases, enzymes, salts and unincorporated nucleotides. 10 volumes o f buffer PN were added and mixed with the reaction sample. Briefly, the mix was applied to a QIAquick column, previously placed in a 2 ml collection tube, and centrifuged for 1 m in at 6000 rpm. The column was washed with 750 pi 80% EtOH/buffer (PE) and centrifuged for 1 min at 6000 rpm. An additional centrifugation for 1 min at 13000 rpm allowed complete removal o f residual EtOH. The DNA was eluted by adding 60 pi elution buffer EB to the column and centrifuging.

2.3.2 Gel electrophoresis

For sizing and separation o f DNA samples, a horizontal submerged agarose gel electrophoresis system was used. The concentration o f agarose (Ultra Pure agarose for electrophoretic grade, Life Technologies) varied according to the size o f the fragments o f interest. For example, 1% gels were used for 500-10"^ bp fragments, 2% for 50-500 bp fragments, 3% for <50 bp. Gels were prepared by dissolving the agarose in Ix TAE buffer (see below), containing ethidium bromide (final concentration o f 100 pg/ml; Sigma Aldrich). The molten agarose was cast in a horizontal tray fitted with well- forming combs, and, once set, was submerged in Ix TAE buffer in the fiat-bed apparatus.

Prior to loading into individual wells, 1/6 total volume o f gel tracking dye was added to the DNA solutions. The samples were electrophoresed at a constant voltage o f 60-100 V, in parallel with an appropriate double-stranded DNA marker (bacteriophage X

digested with Pst I, Life Technologies, or 100 bp DNA ladder, Promega). DNA bands were visualised using a 254 nm transilluminator. Gels were photographed using a Polaroid MP4 Land Camera and Polaroid 667 film type (Sigma Aldrich).

Buffers and solutions:

Tris base 242 _g

Glacial acetic acid 57.1 ml

EDTA (0.5 M, pH 8.0) 100 ml

Distilled water (dHiO) to 1000 ml This buffer can be stored at room temperature (rt).

Gel tracking dve (6x1:

Bromophenol blue 0.25 _g

Sucrose 40.0 _g

EDTA (0.5 M, pH 8.0) 4.0 ml

dHzO to 100 ml

2.3.3 Extraction of DNA from gels

DNA bands were visualised with UV light and cut from the gel using a sterile blade. They were weighed in a 1.5 ml plastic tube, dissolved in three volumes o f Nal solution (B IO -101, Vista, CA) and melted at 55°C. 10-15 pi EZ-glassmilk solution (B IO -101) was added to the molten slice and incubated for 5 min at rt. After centrifugation at 13000 rpm in a microcentrifuge, the pellet was eluted with 25 pi sterile dHiO for 5-15 min at 55°C. The solution was then vortexed, centrifuged and the supernatant was transferred to a new tube and stored at -20°C.

2.3.4 Ligation of DNA fragments into plasmid vectors

A number o f different plasmid vectors were used in this study. They are described with the specific manipulations in the results sections (Chapters 3-7). A general procedure for ligation is outlined here.

Both the insert and the vector were digested with their respective restriction enzymes. The DNA was electrophoresed, specific bands excised and extracted from the gel as described above. To estimate the amount o f DNA extracted, the samples were run on a gel in parallel with markers o f known molecular weight and concentration, and the intensities o f the isolated bands compared under UV light. Dividing the estimated DNA weight by the size o f the fragment, an approximation o f the relative molarities o f both vector and insert was derived. The following reactions were set up: vector and insert at molecular ratios o f 1:2, 1:4, 1:8; vector alone and water, as negative controls.

Ligations were carried out overnight (o/n) at rt in 10 pi total volume, with 1 pi T4 DNA ligase (Life Technologies), and 2 pi 5x ligation buffer (Life Technologies).