2.3.4.1 DNA Isolation from Agarose Gels
DNA extraction from agarose gels was carried out using the ‘Ultrafree-DA Centrifugal Filter Device’ (Millipore).
2.3.4.1.1 Theory
The Ultrafree-DA device from Millipore was designed to recover 100 to 10,000 bp DNA from agarose gel slices in one ten-minute spin. It consists of a pre-assembled sample filter cup with agarose ‘Gel Nebulizer’, and a microcentrifuge vial. The device utilises gel compression to extract DNA from the agarose. Centrifugal force collapses the gel structure, drives the agarose through a small orifice in the ‘Gel Nebulizer’ and the resultant gel slurry is sprayed into the sample filter cup. As the agarose is compressed at 5,000xg, DNA is extruded from the gel's pores. The gel matrix is retained by the microporous membrane, and the DNA passes freely through the membrane. DNA can then be recovered in the filtrate vial.
2.3.4.1.2 Protocol
- Required DNA product was first electrophoresed through a modified TAE agarose gel (refer to section 2.3.2).
- Band of interest was located using a long wavelength UV lamp, and the slice of agarose that contained the DNA was carefully excised from the gel (any excess agarose was also then trimmed away).
- The gel slice was then placed into the ‘Gel Nebulizer’ sample cup assembly device and sealed, so that the cap was attached to the vial.
- The ‘Gel Nebulizer’ and vial were then centrifuged at 5000xg fro 10 minutes.
- DNA was now present in the filtrate. The filter cup and ‘Gel Nebulizer’ were then discarded, allowing the DNA to be stored in the capped filtrate vial.
2.3.4.2 Miniprep Protocol (QIAgen Spin Miniprep Kit – Qiagen)
The ‘QIAgen Spin Miniprep Kit’ was used to isolate all the plasmid DNA screened for the required UCH-L1 promoter inserts. The protocol is described below (all buffers were part of the Qiagen kit):-
- 0.5ml of the overnight culture (above) was centrifuged at 13,500rpm for 45 minutes.
- The bacterial cells were resuspended in 250µl of Buffer P1 (+ RNase A) until no cell clumps were visible.
- 250µl of Buffer P2 was then added. Tubes were inverted 4-6 times to mix.
- 350µl of Buffer N3 was added, and tubes were inverted until the solution became cloudy. - Tubes were then centrifuged at 13,500 rpm for 10 mins.
- Supernatants from the above step were decanted into the ‘QIAprep columns’ which were then centrifuged at 13,500rpm for 1 minute. The flowthrough was discarded.
- The QIAprep spin column was then washed by adding 0.5ml Buffer PB, and the centrifuge step at 13,500rpm for 1 minute was repeated. The flowthrough was again discarded.
- A second wash step was carried out by adding 0.75ml Buffer PE, and again centrifuging for 1 minute at 13,500rpm.
- The flowthrough was discarded, and the column was centrifuged for an additional 1 minute to remove any residual wash buffer.
- The plasmid DNA was finally eluted into a clean microcentrifuge tube by adding 50µl Buffer EB to the centre of the QIAprep column, and centrifuging for 1 minute at 13,500rpm after a 1 minute diffusion period.
2.3.4.3 Midiprep Protocol (‘PureYield’ Plasmid Midiprep System – Promega)
The ‘’PureYield’ Plasmid Midiprep System’ was used to isolate all the plasmid DNA that was required for cloning ligation reactions (refer to section 2.3). The protocol, split into two stages, is described below (all solutions and columns were part of the Promega system):- 2.3.4.3.1 Preparation and Lysis of Bacterial Cell Cultures
- Transformed E.coli bacterial cells were grown up overnight (≈16 hours) in a 50ml culture. - The cells were then centrifuged at 10000 x g for 10 minutes in order to pellet the cells. The supernatant was then discarded, any excess media was drained on a paper towel.
- The cell pellet was then completely resuspended in 3ml of ‘Cell Resuspension Solution’. - 3ml of ‘Cell Lysis Solution’ was then added and gently mixed by inverting the tube four times. The tube was then incubated for 3 minutes at room temperature.
- 5ml of ‘Neutralization Solution’ was then added to the lysed cells, which was then capped and gently inverted four times to mix. The tube was then left in an upright position for 3 minutes to allow a white flocculent precipitate to form.
2.3.4.3.2 DNA Purification by Centrifugation
- A ‘PureYield Clearing Column’ was first placed into a new 50ml disposable plastic tube. - The lysate (from stage 1 above) was then poured in and left to stand for 2 minutes, so as to allow any cellular debris to rise to the top.
- The ‘PureYield Clearing Column’ was then centrifuged at 3000 x g for 5 minutes to filter the lysate through (the centrifugation step was repeated if deemed necessary).
- A ‘PureYield Binding Column’ was then placed into a new 50ml disposable plastic tube. - The filtered lysate was then poured in and centrifuged for 3 minutes at 1500 x g.
- 5ml of ‘Endotoxin Removal Wash Solution’ (+ Isopropanol) was then added to the ‘PureYield Binding Column’ and centrifuged at 1500 x g for 3 minutes. The flowthrough was then discarded, and the column reinserted into the tube.
- 20ml of ‘Column Wash Solution’ (+ ethanol) was then added to the ‘PureYield Binding Column’, and centrifuged at 1500 x g for 5 minutes. The flowthrough was then once again discarded, and the column reinserted into the tube. Centrifugation at 1500 x g was then carried out for an additional 10 minutes to ensure complete ethanol removal from the column (the tip of the column was also tapped on a paper towel to remove any final traces).
- The DNA was then eluted by placing the binding column into a new 50ml disposable plastic tube, and adding 600µl of ‘Nuclease-Free Water’ to the binding membrane of the ‘PureYield Binding Column’.
- The ‘PureYield Binding Column’ was then centrifuged for 5 minutes at 1500 x g. The filtrate was then finally transferred into a new 1.5ml tube.