Extraction of Plasmid DNA

After the overnight culture the bacteria were pelleted by centrifugation at 3500rpm for 10 minutes. Excess medium was carefully pipetted off and the pellet resuspended in 250pl of resuspension buffer P1(100pg/ml RNase A, 50mM Tris/HCI, lOmM EDTA, pH 8.0). This was then transferred to a 1.5ml microcentrifuge tube (Eppendorf) and 250pl of buffer P2 (200mM NaOH, 1%SDS) was added, which denatures cellular proteins and chromosomal DNA. The suspension was gently inverted to avoid shearing of chromosomal DNA and incubated for 5 minutes at room temperature. 350pl of chilled buffer N3 (3.0M KC2H3O2, pH 5.5) was added to the mixture, which results in precipitation

of the denatured proteins, chromosomal DNA, cellular debris and SDS, while allowing the shorter plasmid DNA to stay in solution. The samples were then centrifuged at 1 0 , 0 0 0 x g in a benchtop microcentrifuge for 1 0 minutes to pellet

the precipitate along the side of the tube. The supernatant was aspirated and applied to a QIAprep-spin column. The column was centrifuged for 1 minute at 10,000 X g and the flowthrough fraction was discarded. In the column is an

anion-exchange resin containing positively charged Diethylaminoethanol groups which bind to the negatively charged phosphate groups of the DNA backbone. Binding and elution of the DNA is determined by the pH and salt concentration of the buffers used. The column was washed with 500pl of wash buffer PB (proprietary formulation), which was then centrifuged at 10,000 x g in the benchtop microcentrifuge. Again the flowthrough fraction was discarded and 750pl of buffer PE (lOmM Tris/HCI pH 8.0, 80% Ethanol) was added to wash the column which was centrifuged at 1 0 , 0 0 0 x g in the microcentrifuge.

The flowthrough fraction was discarded and the column was recentrifuged at 10,000 X g for a further 1 minute to remove any residual wash buffer. The QIAprep-spin column was then placed in a fresh 1.5ml microcentrifuge tube (Eppendorf) and the DNA was eluted by the addition of lOOpI of distilled H2O to

the column ensuring the resin was completely covered. The column was spun at 10,000 X g for 1 minute and the plasmid DNA collected in the 1.5ml

microcentrifuge tube.

2.2.3.1 Restriction Digestions

Approximately Ijag of each of the plasmid DNA was incubated with 2|liI of the appropriate lOx restriction enzyme buffer, 10 lU of the desired restriction enzyme, bovine serum albumin (BSA) to a final concentration of 10Opg/ml if required and sterile H2O to a final volume of 20pl. The mixture was incubated

for 2 hours at 37°C. To visualise the digested DNA 2|il of each digest was added to 2\i\ of DNA loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water) and 6 pl H2O and loaded onto a 0.8% agarose

gel (section 2.2.1.2). The gel was run at 5 volts/cm in 0.5x TBE and the DNA visualised using a UV light box.

1x concentration of restriction enzyme buffers used:

Promega Buffer B (pH 7.5, 6 mM Tris-HCI, 6 mM MgCl2, 50mM NaCI, ImM

DTT); {Nhe I / Spe I)

Promega Buffer D (pH 7.9, 6 mM Tris-HCI, 6mM MgCl2, 150mM NaCI, ImM

DTT): (Sal I)

Promega Buffer G (pH 8.2, 50mM Tris-HCI, 5mM MgCl2): (Miu Nl)

NEB buffer 2 (pH 7.9, 10 mM Tris-HCI, lOmM IVIgClg, 50 mM NaCI, ImM DTT):

{Hind III / Xba I + BSA / Xho \ + BSA / Eco RV + BSA)

NEB buffers (pH 7.9, 50mM Tris-HCI, lOmM MgClg, lOOmM NaCI, ImM DTT):

(Bgl II / Not\ + BSA / Dra III + BSA)

NEB buffer 4 (pH 7.9, 20mM Tris-acetate, lOmM Magnesium acetate, 50mM potassium acetate, ImM DTT): (Sna Bl + BSA / Sma I)

BamHI buffer (NEB(pH 7.9, 150mM NaCI, lOmM Tris-HCI, lOmM MgCl2, ImM

dithiothreitol) supplemented with BSA lOOpg/ml)

Nsi I buffer (NEB(pH 8.4, lOmM Tris-HCI, lOmM MgCl2, lOOmM NaCI, ImM

DTT))

Sal I buffer (NEB(pH 7.9, 150mM NaCI, 10mM Tris-HCI, lOmM MgCl2, ImM

dithiothreitol) supplemented with BSA lOOpg/ml)

Boehringer Buffer H (pH 8.0, lOmM Tris-HCI, 5mM MgCl2, lOOmM NaCI, ImM

2-Mercaptoethanol): (Pvu I)

2.2.3 2 Extraction of DNA From 0.8% Agarose Gel

After restriction digestion and prior to ligation, DNA fragments may require separation which was achieved by running the DNA on an agarose gel. The DNA was visualised using a long wavelength UV lamp to minimise damage to the digested DNA and the desired DNA band was cut out, minimising the surrounding area of agarose and transferred to a 1.5ml microcentrifuge tube. Each gel fragment was weighed and the DNA extracted from the gel using the Qiagen Qiaquick gel extraction kit. To the sample was added lOOpI Buffer QX1 (proprietory formulation - solubilises the agarose) per lOOmg agarose and lOpI of QIAEX II (proprietory formulation - DNA absorbs to silica particles) and the

samples mixed by pipeting up and down (Vogelstein and Gillespie, 1979). The sample was then incubated at 50“C for 10 mins, gently agitating the tubes 2-3 times during this period to aid mixing of the solution. The sample was centrifuged at 1 0 , 0 0 0 x g in a microcentrifuge for 1 minute and the supernatant

removed with a pipette. The sample was resuspended in 500pl Buffer QX1 and recentrifuged at 1 0 , 0 0 0 x g in a microcentrifuge and the supernatant again

removed with a pipette. SOOpI of Buffer PE was added to the tube, the pellet resuspended and sample recentrifuged at 10,000 x g. This step with Buffer PE was repeated one more time as described above and after careful removal of the supernatant the pellet of QIAEX II bound to the DNA fragments was allowed to dry for 15 minutes at room temperature. To elute the DNA 20pl of H2O was

added to the sample and the pellet resuspended by briefly vortexing. The sample was again centrifuged at 1 0 , 0 0 0 x g for 1 minute and the supernatant

containing the eluted DNA was transferred to fresh 1.5ml microcentrifuge tubes.

2 2.3.3 Ligation

Ligation of vector and insert DNA fragments was performed using T4 DNA ligase, which catalyses the joining of two DNA strands between the 5'- phosphate and 3’-hydroxyl groups of adjacent nucleotides in either a blunt- ended or cohesive-end configuration. The vector and the insert DNA at a ratio of 1:5 were added to the ligation mix consisting of polyethylene glycol at a final concentration of 5%, 50mM Tris-HCI (pH 7.5), lOmM IVIgClg, lOmM dithiothreitol, ImM adenosine triphosphate (ATP), 25pg/ml bovine serum albumin and 100 units of T4 DNA ligase (NEB). As an indicator of the ability of the linearised vector to religate an aliquot of the linear vector was incubated

with the ligation mix and to assess the presence of uncut vector contaminating the linearised sample of vector plasmid an aliquot of the linearised vector was incubated in the absence of the ligase mix. All the above mixtures were then incubated at 16°C overnight and then 25pl of each of the three mixes were transformed into competent cells (see 2.2.2.1), which were then selected on LB agarose plates containing the appropriate antibiotics.

2.2.3.4 Cleaning of Plasmid DNA Using Qiagen Clean up Column

Plasmid DNA was purified prior to restriction digestion, ligation or transfection to remove substances that may interfere with these processes. The purification process is based on binding of the plasmid DNA to a silica membrane, which is enhanced at a high salt concentration (Vogelstein and Gillespie, 1979). To the plasmid DNA was added 10 times the volume of the DNA sample to be cleaned of buffer PN (proprietory formulation - enhances binding of DNA to silica gel due to high concentration of chaotropic salt). The DNA mix was then loaded onto a clean up column, containing the silica membrane, which was placed in a 2ml microcentrifuge tube provided. The sample was spun at 5000 x g in a benchtop microcentrifuge for 1 minute at room temperature. The flow through fraction was discarded and 750pl of wash buffer PE was added to the column, which was then respun at 5000 x g in the microcentrifuge for 1 minute at room temperature. The eluate was again discarded and the column was respun at 1 0 , 0 0 0 x g for 1 minute in the

microcentrifuge to remove any residual Buffer PE. The DNA was then eluted by pippetting 50^1 of distilled HgO, taking care to cover the entire membrane and allowing the column to stand for 1 minute at room temperature. The

column was placed in a fresh 1.5ml microcentrifuge tube (Eppendorf) and both were spun at 10,000 x g for 1 minute at room temperature to elute the DNA into the microcentrifuge tube.