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DNA, like many biological molecules, carries an electrical charge which in this case is negative. If DNA is loaded into an agarose gel and an electrical field is applied, the DNA will migrate through the gel towards the positive electrode. This is the principle

o f DNA electrophoresis. There are a great number o f factors which influence

electrophoretic mobility in gel media and, in the case o f DNA, size is the principal factor. DNA migrates through pores in the gel matrix at a rate inversely proportional to size.

Typical agarose concentrations for gels fall within the range 0.3 - 2.0% (w/v), the concentration used depends on the predicted sizes o f nucleic acids to be separated

(Sambrook et aL, 1989):

Linear nucleic acid size (kb) % Agarose

0 .1 - 5 2 . 0 0.2 - 6 1.5 0 .3 -1 0 1 . 0 0.4 - 20 0 . 8 0.7 - 45 0.5 1 .0 -7 0 0.3

For cosmids and PAC restriction digests with EcoRI, agarose gels o f 0.8% were prepared.

For digestion o f plasmid insert from vector, agarose gels o f 1.0% were prepared.

For checking o f size and concentration o f Polymerase Chain Reaction (PCR) products, agarose gels o f 1.5 - 2.0% were prepared.

To view the progress o f DNA present in an agarose gel the gel needs to be stained with 0.5 mg/ml ethidium bromide prior to electrophoresis. Ethidium bromide intercalates between adjacent base-pairs in DNA and fluoresces under ultraviolet (UV) light. This enables the gel to be photographed with the aid o f a UV transilluminator.

3.2.1 Preparation of agarose gels

The gel tray was prepared for the addition o f molten agarose by sealing both ends with masking tape and placing a clean, well-forming comb into position.

3 different gel trays (GIBCO-BRL) were used depending on the size o f the gel required:

Size Volume o f molten agarose

mini (5.7 x 8.3cm) 25ml

midi ( 1 1 X 14cm) 1 0 0ml

maxi (20 x 25cm) 350ml

For a 0.8% midi agarose gel, 0.8g o f agarose was added to 100 ml Ix TBE buffer and dissolved by boiling. The molten agarose was cooled to approximately 50 ° C, when 5pl o f ethidium bromide (lOmg/ml) (BDH) was added. The molten gel was then poured into the gel tray avoiding the formation o f air bubbles which impede the migration o f DNA through the gel. After allowing the gel to set at room temperature the tape was removed and the gel tray was placed into an electrophoresis tank. Ix TBE buffer was

poured into the tank until the gel was submerged by about 1mm and the well-forming

comb removed ready for the addition o f DNA samples.

3.2.2 Preparation of DNA samples for electrophoresis

DNA samples were mixed with a 5x loading buffer to give a Ix final concentration. This loading buffer consists o f 15% (w/v) ficoll which sinks the DNA samples into the wells formed within the gel. It also consists o f 0.25% (v/v) bromophenol blue and 0.25% (v/v) xylene cyanol which act as tracker dyes allowing the progress o f the DNA to be followed as it is electrophoresed.

By including DNA fragments o f known molecular weight (MW) on a gel during electrophoresis, the size o f a DNA fragment can be determined by its migration through

commercially and two commonly used DNA markers were a Ikb DNA ladder (GIBCO- BRL) and a lOObp DNA ladder (GIBCO-BRL).

The Ikb DNA ladders’ markers range from 298bp - 12.216kb and is so called as it is prepared from a plasmid containing 12 repeats o f a l.OlSkb DNA fragment in addition to vector fragments that act as reference markers to DNA fragments under 2.036kb. The lOObp DNA ladders’ markers range from lOObp - 2.072kb and is so called because it consists o f 15 repeats o f a lOObp DNA fragment and an additional 2.072kb vector DNA fragment. For both ladders Ip g o f DNA was added per lane.

3.2.3 DNA electrophoresis

The voltage and time for which a gel is electrophoresed is dependent on the quality o f result required. To simply confirm the presence o f DNA in the sample the gels were electrophoresed at lOOV in Horizontal Gel Electrophoresis tanks (GIBCO-BRL) (Section 2.6) until the samples were visible or the size approximated. To run a gel for accurate size determination o f digested DNA fragments or for eventual Southern blotting the gel was run at a lower voltage 0 /N until the bromophenol blue had migrated

3/4 distance. The voltage used was dependent on the length o f the gel, but an

approximate guideline was 2V/cm. After electrophoresis, the gel was photographed w ith the aid o f a UV transilluminator. A flourescent ruler was laid down the side o f a gel to be Southern or northern blotted (Section 3.3.3) to allow DNA/RNA size identification from a resulting autoradiograph.

3.2.4 Recovery and purification of DNA fragments from agarose gels using the Qiagen QIAquick Gel Extraction Kit

After DNA electrophoresis, DNA fragments o f interest can be excised from agarose gels and purified. They can then be labelled and used as probes in hybridisation experiments or subcloned and the DNA sequenced.

The Qiagen QIAquick Gel Extraction kit provides all its own buffers and apparatus and is designed to extract and purify DNA from TBE agarose gels with high percentage yields.

The DNA fragment was excised from the gel using a sharp, sterile scalpel. The gel slice was weighed and a 3x volume o f buffer Q X l was added to the gel slice. The gel slice was dissolved into the buffer at 50 °C for lOmins, then placed into a QIAquick spin column. The spin column was centrifuged for 60 secs and the flow-through fraction discarded. The spin column was then washed with 750pl o f buffer PE and centrifuged for 60 secs. Again, the flow-through fraction was discarded and the spin column

centrifuged for a further 60 secs. The spin column was then placed into a clean

eppendorf and the bound DNA eluted with 50pl TE pH 8.0. The concentration o f the purified DNA fragment was estimated by electrophoresing 5pi on an agarose gel.