2.1.1 Restriction enzyme digestion
Restriction enzyme digests were conducted using Fast Digest enzymes in a total volume of 20 µl.
Each reaction contained 1 µl Fast Digest Enzyme (Thermofisher, South Africa), up to 1 µg plasmid DNA, 2 µl of 10x Fast Digest Green Buffer and dH2O to make up the remaining volume. The reactions were incubated at 37 ˚C for 60 minutes to allow for optimum enzyme activity. In some cases, negative controls were included where the restriction enzyme was omitted.
2.1.2 Separation of DNA fragments by agarose gel electrophoresis
DNA fragments were subjected to agarose gel electrophoresis to separate fragments according to their molecular weight. An aliquot of 2 µl 10X Fast Digest buffer green was added to the sample prior to electrophoresis. Agarose gels were made up to 1% using SeaKem LE agarose (Lonza, South Africa) and 1 x TBE, which was supplemented with 0.5 µg/mL ethidium bromide (Merck, South Africa) to enable visualization of the DNA under UV light. The DNA was resolved on an agarose gel, suspended in 1 X TBE buffer, for 45 minutes at 100 V. A 10 µl aliquot of GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific, South Africa) was resolved alongside the experimental samples to allow for estimation of the DNA band sizes. Agarose gels were viewed on the Gel Doc Go system (Bio-Rad, South Africa) and the images were captured using Image Lab 6.1 Software (Bio-Rad, South Africa).
2.1.3 Excision and recovery of DNA fragments from agarose gels
The bands of interest were excised from the gel with a scalpel using the Accuris SmartBlue Blue Light Transilluminator (Accuris Instruments, USA) to avoid DNA damage which could occur when using UV light for DNA visualisation. The DNA was then purified using the QIAquick Gel Extraction Kit (Qiagen, South Africa) according to the manufacturer’s instructions. The gel slices were transferred to Eppendorf tubes and then weighed. Three volumes of Buffer QG were added to the agarose gel (volume:weight) before incubation at 50 ˚C for 10 minutes, to completely
53
dissolve the agarose. The tubes were vortexed every 2-3 minutes to mix the contents. The colour of the mixture was confirmed to be yellow before proceeding as this indicative of the pH of the solution. The ideal pH for DNA binding to the column matrix is pH ≤ 7.5, which is signalled by the Buffer QG turning yellow at pH ≤ 7.5. One gel volume of isopropanol was added to the sample which was then applied to the Qiaquick spin column. The column containing the sample was centrifuged at ≥ 10 000 x g for 1 minute to pass the solution containing DNA over the column matrix. The flow through was discarded and 0.75ml Buffer PE was added to the column. The sample was centrifuged again, for 1 minute, and the flow through was discarded. This was followed by an additional 1 minute centrifuge spin to remove any residual wash buffer. An aliquot of 30 µl pre-warmed Buffer EB was added to the centre of the Qiaquick membrane to elute the DNA, which was bound to the column matrix. The column was incubated with the buffer for 5 minutes before repeating the centrifugation step to elute the DNA. DNA was either used immediately or stored at -20 ˚C.
2.1.4 Ligation of DNA fragments
The digestion of plasmid DNA yielded sticky ends, that were compatible between the plasmid backbone and the insert. Ligations were conducted using the Quick LigationTM Kit (New England BioLabs, South Africa). A 3:1 molar ratio of insert to backbone was used in a reaction volume of 20 µl. This comprised of 1 µl Quick Ligase (New England BioLabs, South Africa), 10 µl 2X Quick Ligase Reaction Buffer, insert, and vector backbone DNA. Controls were included where there was either 1) no insert DNA to assess self-ligation, 2) no insert DNA and no ligase to determine the prevalence of residual undigested DNA. Reactions were incubated at room temperature for 30 minutes.
2.1.5 Transformation of Escherichia coli
Escherichia coli (E.coli) DH10 chemically competent cells (Inqaba Biotechnology, South Africa) were transformed with an aliquot of 0.1 - 1 ng of plasmid, or 1.5 µl ligation reaction. After thawing the cells on ice, 15 µl of competent E.coli cells were added to an Eppendorf tube containing the plasmid DNA. The contents were gently mixed by pipette aspiration. The tubes were incubated on ice for 20 minutes and then heat shocked at 42 °C for 30 seconds. The samples were then returned
54
to ice for a further 2 minutes. An aliquot of 400 µl of SOC Outgrowth Medium (New England BioLabs, USA) was added to the cells and the samples were incubated at 37 °C on a shaker, for 60 minutes, to allow for expression of the antibiotic selection genes. Aliquots of 25 µl, 50 µl and 150 µl of each sample were plated onto LB agar plates (Appendix A), supplemented with 25 µg/ml Chloramphenicol (Sigma-Aldrich, South Africa) for pSSPEx vectors and 100 µg/ml Carbenicillin for pMEx plasmid DNA. E.coli transformants containing the plasmids were grown in broth culture, with appropriate selection, and then stored as 25% glycerol stocks at -80 ˚C.
2.1.6 Isolation of DNA
Plasmid DNA for transfections was isolated from the E.coli cells using the QIAGEN® Plasmid Plus Maxi Kit. Small scale plasmid DNA isolation was done for screening putative clones using alkaline lysis.
2.1.6.1 Large scale isolation of plasmid DNA
Plasmid DNA was isolated using the QIAGEN® Plasmid Plus Maxi Kit (Qiagen, USA) in accordance with the protocol described for high yield DNA. Recombinant E. coli cells were propagated in 100 ml liquid media for large scale plasmid isolations. The bacterial culture was harvested by centrifuging at 6000 x g for 15 minutes at 4 °C and the pellet was resuspended in 8 ml Buffer P1. An aliquot of 8 ml Buffer P2 was added, and the tube was gently mixed until the contents became viscous. The sample was incubated at room temperature for 3 minutes to enable cell lysis to occur. Subsequently, 8 ml of Buffer S3 was added to neutralize the lysate and the solution was mixed by inverting 4-6 times. The lysate was then transferred to the QIAfilter cartridge and incubated at room temperature for 10 minutes to allow the cell debris to settle. While incubating the lysate, the QIAGEN Plasmid Plus spin column was placed into the QIAvac 24 Plus and Tube Extenders inserted. The plunger was gently inserted into the QIAfilter cartridge, and the lysate was filtered into a new tube enabling the removal of undesired cell debris. An aliquot of 5 ml binding buffer (BB) was added to the cleared lysate and inverted 4-6 times to mix. The addition of buffer BB promotes the binding of DNA to the spin column to maximise recovery of the recombinant plasmid. The lysate was transferred to the QIAGEN Plasmid Plus spin column, with a tube extender attached on the QIAvac 24 Plus. Approximately -300 mbar vacuum was applied until the liquid had been drawn through the column. The bound DNA was sequentially washed
55
with 0.7 ml endotoxin removal buffer (ETR) and 0.7 ml wash buffer (PE) which were drawn through the tube by application of a vacuum. The QIAGEN Plasmid Plus Maxi spin column was placed into a 2 ml collection tube and centrifuged at 10 000 x g for 1 minute to remove any residual wash buffer. The QIAGEN Plasmid Plus spin column was then placed into a clean 2 ml tube and 400 µl prewarmed elution buffer (EB) was added to the centre of the QIAGEN Plasmid Plus spin column. The column was incubated for 5 minutes and then centrifuged for 1 minute to elute the DNA. Plasmid DNA was stored at 4 ˚C.
2.1.6.2 Small scale isolation of plasmid DNA
Alkaline lysis was used to extract DNA from cells. A 12-well plate was simultaneously seeded with 0.1 x 106 RK13 cells and infected with recombinant virus. The infection was incubated at 37˚ C under standard cell culture incubator conditions for 72 hours. After 72 hours, the media was discarded and the cells were resuspended in 250 µl solution P1 (Appendix A) which contains glucose to maintain osmotic pressure outside the cells, Tris for maintenance of pH and EDTA to protect the DNA from degrading. An aliquot of 250 µl solution P2 (Appendix A) was added to lyse the cells by pipetting the mixture over the cells several times. The mixture was transferred to a 1.5 ml Eppendorf tube and 350 µl solution P3 (Appendix A) was added to neutralize the samples.
The tube was gently inverted 4-6 times to ensure complete neutralization resulting in the formation of white precipitation of DNA and protein. The samples were spun at max RPM for 10 minutes on a benchtop centrifuge to separate the DNA and proteins; and 750 µl of clear supernatant was pipetted out into new 1.5 ml Eppendorf tubes. The DNA at this point was in the supernatant. An aliquot of 750 µl ice cold isopropanol was added to precipitate the DNA out of solution, and the samples were stored at -20˚ C for 1 hour. Samples were subsequently centrifuged on a benchtop centrifuge at maximum speed for 20 minutes. The isopropanol was carefully discarded so as not to disturb the pellet containing the DNA. To remove any residual salt, an aliquot of 70% ethanol was added, and the samples centrifuged for 1 minute at max speed. The ethanol was also carefully removed, and the pellet left to air dry for 5 minutes before being resuspended in 50 µl RO water.
56