DNA transformation and isolation

2.8.1 Preparation of LB agar plates and LB broth

LB agar plates were prepared by dissolving 12.5 grams of Luria-Bertani (LB) (Invitrogen, UK) powder in 500 ml of distilled water followed by addition of 7 grams of agar (Sigma, UK). The solution was then autoclaved at 15psi and 121oC. The solution was then left to cool down under aseptic condition and ampicillin (100 µg/ml) was added. Approximately 10ml of the cooled mixture was poured in sterile 90cm petri dishes and stored at 4oC.

76 LB broth was prepared by adding 12.5gr of LB into 50ml of distilled water which was then autoclaved. After the solution was cooled down ampicillin (100 µg/ml) (Sigma, UK) was added to the solution under aseptic conditions.

2.8.2 Transformation

The process of bacterial cells taking up naked DNA molecules is called transformation. Since the bacterial genome is made up of circular DNAs and plasmids it has the ability to replicate foreign DNA alongside their own DNA when the foreign plasmid DNA has an origin of replication recognized by the host cell DNA polymerases.

Per transformation 20 µl of DH5α strain of Escherichia coli (E. coli) (Invitrogen, UK) were thawed on ice, 200 ng of DNA was added to the cells and carefully mixed by flicking the tube. Then cells were left on ice for 30 minutes and then heat shocked at 42oC for 45 seconds then placed on ice for 2 minutes. 500 µl of LB broth was added to the cells and they were incubated in a shaker for 1 hour at 37oC. 200 µl of the resultant mixture was streaked on an LB agar plate containing ampicillin. Then the plate was incubated for 16 hours at 37oC.

2.8.3 Plasmid purification: maxi preps

Plasmid DNA that was obtained from DH5Aα (Invitrogen, UK) cells colonies through transformation was inoculated in LB-Amp overnight at 37° C with shaking. The samples were centrifuged at 4000g for 10 minutes and the supernatant was discarded. Maxi preps were carried out using the protocol and buffers provided with the Invitrogen Maxi prep Kit. First the filtration cartridge was inserted in the PureLink HiPure Maxi Column. Then 15ml of Equilibrium Buffer (EQ1) were directly added to the filtration catridge, the solution was left to drain by the gravity flow. Next 10 ml of resuspension buffer (R3) with RNAase A were added to the pellet until the cell suspension became homogenous. Next 10ml of lysis buffer (L7) was added to the pellet and left at room temperature for 5 minutes until the mixture became homogenous. Then 10 ml of precipitation buffer (N3) was added, in order to mix the solution, the tubes were inverted. Finally, the precipitated

77 lysate was transferred into the column, the solution was left to flow through the column by gravity . The inner cartridge was then discarded, and the column was washed with 50 ml Wash Buffer (W8). A 50 ml centrifuge tube was placed underneath the HiPure Filter Column. 15 ml of elution buffer (E4) were added to the column, again the solution was left to follow through column by gravity. The DNA was then precipitated with isopropanol and collected by centrifugation at 15, 000 rpm at 4 °C for 30 minutes. The pellet was then washed with 70% ethanol, and then the pellet was resuspended in 300 µl of TE buffer. The concentration of DNA was then measured using 1 µl of the maxi prep on a Nanodrop at 260 nm.

2.8.4 Plasmid purification: mini prep

A single colony from the agar plate was inoculated in 3ml of LB containing ampicillin and left in a 37 °C shaker for 15 hours. The DNA was then purified using the mini prep kit from Bioline, following the manufacturers guidelines.

The inoculation was centrifuged for 30 second at 11,000g, and the supernatant was discarded. Pellet was re-suspended in 250 µl of P1 buffer by vortexing, then a further 250 µl of lysis buffer P2 were added to the pellet. The solutions were then mixed by gently inverting the tubes 6 to 8 times. Then the tubes were left at room temperature for 5 minutes until the lysate appears clear. 300 µl of neutralization buffer P3 was added and then the lysate was centrifuged at 11,000 g for 5 minutes. The ISOLATE II Plasmid Mini spin column was place onto a 2ml collection tube. Then 750 µl of the clarified sample was pipetted onto the column. The flow-through was discarded and the solution was centrifuged at 11,000 g for 1 minute. 500µl of PW1 buffer that was preheated at 50°C was then added to the pellet and the lysate were centrifuged at 11,000g for 1 minute. Then 600µl of PW2 buffer was added followed by centrifugation for 1 minute at 11,000g was carried out. The flow-through was discarded at this point, and the collection tube was centrifuged for 2 minutes at 11,000g. 50µl of elution buffer P was directly added onto centre of silica membrane, and was left at room temperature for 1 minute. A final centrifugation step for 1 minute at 11,000g was carried out and then the concentration of DNA was measured using 1 µl of the mini prep on a Nanodrop at 260 nm.

78 2.8.5 Measurement of DNA concentration

Determining nucleic acid concentration can be achieved by UV light as the nucleic acids absorb the UV light. UV light also is used to measure the purity of the DNA. DNA yield determination is based on its ability to absorb light at maximally 260nm. Presence of excess salt, contaminating proteins or organic solvents may affect DNA purity, the ratio of the absorbance at 260nm to 280nm provides an estimation of the purity. For the DNA sample to be considered pure the ratio should be close to 1.8±0.05. The DNA concentration and its purity was determined using NanoDrop ND-1000 UV visible spectrophotometer. Instrument was calibrated using 1 µl of distilled water, after the calibration step 1 µl of sample was loaded to measure the concertation of desired sample.