DNA Manipulation

2.2.1 Standard Polymerase Chain Reaction (PCR)

Standard PCR was used to amplify DNA fragments for cloning, diagnostic or semi- quantitative analysis (for example, diagnostic PCR of recombinant plasmids during cloning process) PCR reactions were set up using 100-500ng DNA sample, 1X Abgene buffer, 1.5mM MgCl2, 0.2mM dNTP, 500nM forward primer, 500nM reverse primer and 1.25 Units of GoTaq (Promega UK) Taq polymerase, in a final volume of 50µl. Typical thermocycling parameters were 1 cycle for 94°C; 25-30 cycles 94° C for 45 seconds, 59°C for 45 seconds; 72°C for 45 seconds; and a final extension step of 72°C for 5 minutes.

2.2.2 Agarose gel electrophoresis 1X loading buffer [40% (v_/

v) glycerol, 60% (v/v) TE Buffer (Tris Ethylene diamine tetra-acetic

acid (EDTA), 10 mM Tris-HCL, 1mM EDTA, pH 8.0] and 1X bromophenol blue were added to the DNA samples of interest. Samples were then pulse-spun in a centrifuge (Eppendorf Minifuge, 20 seconds, 12,000 rpm) and loaded onto the appropriate percentage agarose gel in 1X Tris-Acetate EDTA (TAE) buffer (400mM Tris-HCL, 20mM glacial acetic acid, 0.1mM EDTA, pH8.0) and run alongside an appropriate sized marker (Promega UK, 100bp and 1kbp). The gel contained ethidium bromide added at a concentration of 0.035µg/ml. The gel was typically electrophoresed in 1X TAE buffer for 1 hour at 80 V. The gel and associated

migrated bands were then visualised on ultraviolet light using a gel documentation and imaging system (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK).

2.2.3 DNA extraction and purification from agarose gel

The required electrophoresed DNA fragments were extracted with a scalpel from the agarose gel and purified using the QIAquick gel extraction kit (Qiagen Ltd), as described in the suppliers handbook. Purified DNA was typically quantified and stored at -20°C.

2.2.4 DNA determination

DNA concentration was determined using the NanaDropTM (ND-1000, Fisher Scientific UK Ltd, Loughborough, UK) spectrophotometer, with absorbance measured at wavelengths of 260nm and 280nm. The DNA purity was determined by the ratio of the absorbance at 260nm/ 280nm.

2.2.5 Ligation of isolated DNA into vector

The appropriate amount of insert DNA was placed into the ligation reaction with 100ng of the vector (pGEM-T Easy, Promega UK). Typically the appropriate amount of insert is calculated so that there was a 3x insert:1x vector ration; (vector, ng × insert sixe, kb)÷(size of vector, kb × insert:vector ration)= ng of insert. The reaction also contained 3U of T4 DNA Ligase (Life Technologies Ltd), 1X TD DNA ligase buffer and H20, to make final volume 10µl. A control reaction is also established where the insert DNA fragment is omitted, thereby allowing one to determine the presence of re-circularised vector plasmid. Both reactions were incubated overnight at 4°C.

2.2.6 Transformation of bacteria with plasmid DNA

Typically 5µl of the ligation reaction was added to 100µl DH5α chemically competent cells (Life Technologies Ltd), mixed gently and incubated on ice (or at 4°C) for 30 minutes. Heat shock treatment at 37°C for 45 seconds was executed using a pre-calibrated water bath. The reaction was then allowed to recover on ice for 2 minutes prior to the addition of 400µl pre-warmed SOC, a high nutrient broth media. The reaction was then incubated for exactly 1 hour at 37°C in a rotating incubator at 225 rpm. Following this 1-hour incubation step, half of the reaction volume was extracted and spread onto a Luria broth (LB) agar plates containing the appropriate selection antibiotic for transformed cell selection (eg. Ampicillin 100µg/ml). Where blue/white selection of transformants was necessary, 40µl X-Gal (20mg/ml) was added to the LB agar plates. Plates were inverted and incubated for 16 hours at 37°C.

Subsequently, single isolated transformant colonies were picked and grown in 10ml of LB (containing the appropriate selection antibiotic) for 16 hours in a rotating incubator at 37°C, at 225 rpm. Using the QIAprep Spin Miniprep kit (Qiagen Ltd) according to the manufacturers instructions, plasmid DNA for diagnostic digest was extracted from 6ml of the 16 hour growth culture. For positive clones, their remaining 4ml of culture was used to make a 20%(v/v) glycerol stock for long-term storage at -80° C, and the remaining 10-15ml was used to establish a secondary culture to extract plasmid DNA for the purpose of further cloning, sequencing or transfection. The plasmid DNA was extracted using the Qiagen Plasmid Mini Kit, following the manufacturer’s instructions.

2.2.7 Restriction enzyme digest

Plasmid DNA was digested using 10U of the required restriction endonuclease (New England Biolabs (UK) Ltd) in a final volume of 20µl, which also contained 1X appropriate restriction endonuclease buffer (Life Technologies Ltd). The digests were incubated for 2

hours at 37°C therefore ensuring complete digestion. Double digests were set up in compatible buffers where appropriate.

2.2.8 Plasmid modification by annealed oligonucleotide cloning

Mutations were introduced into plasmid sequences (for example, to determine functionality of predicted regulatory DNA sequences) through cloning of complementary annealed oligonucelotides. Oligonucleotide sequences were designed using Primer3 primer design software (http://primer3.ut.ee/), to predict the formation of hairpins and loops that would prevent successful annealing. Complementary oligonucleotides, with overhangs to allow subsequent directional cloning, were purchased from Life Technologies Ltd.

Oligonucleotides (1ug of each, in equimolar concentrations) were then resuspended in 50uL annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA). After heating to 90-95°C for 3-5 minutes, the samples were allowed to slowly cool to room temperature (~45 minutes). Annealing was confirmed by agarose gel electrophoresis as in section 2.2.2, although the gel was visualised using methylene blue staining rather than UV irradiation to eliminate the possibility of DNA damage for subsequent experiments. Methylene blue staining was performed in 0.002% methylene blue (w_/

v, Sigma-Aldrich M-4159) solution in 0.1X TAE

(0.004M Tris 0.0001 M EDTA) for 1 hour at room temp. DNA extraction and ligation was subsequently performed as in 2.2.3-2.2.5.