General cloning techniques

2.3.7.1 Production of competent bacteria

Competent cells were produced using a magnesium chloride/calcium chloride method(Sambrook and Russel 2001). Non-competent E.coli, strain JM109 (Promega), were subcultured in 4 ml Luria Bertani (LB) medium overnight, 37oC. Four millilitres of the overnight culture was inoculated into 400 ml LB medium, which was incubated with regular measurements made of the optical density using a SmartSpec™ Plus spectrophotometer (BioRad). When the OD600 reached 0.4, cells were split into eight, 50 ml polypropylene tubes and cooled on ice for 10 minutes. The cultures were centrifuged at 2700 x g, 10 minutes, 4oC and the supernatant removed. Each cell pellet was resuspended by swirling with 30 ml MgCl-CaCl solution, 4oC, before being centrifuged as above. Each cell pellet was resuspended in 2 ml CaCl solution at 4oC, following which all eight suspensions were pooled. Five hundred and sixty microlitres of dimethyl sulfoxide (DMSO) was added to the 16 ml of suspension, which was incubated on ice for 15 minutes. A further 560 µl of DMSO was added and 100 µl aliquoted into pre chilled polypropylene tubes. Cells were stored at -80oC. 2.3.7.2 Transformation of competent bacteria

Competent E. coli were thawed on ice. Twenty five to fifty microliters of thawed bacteria were transferred to polypropylene tubes containing 1 µl (1-3 µg) of plasmid and incubated on ice for 30 minutes. Cells were then heat shocked in a water bath at 42oC for 45 seconds before being returned to ice for 2 minutes. Each culture received 850 µl SOC medium and was then incubated at 37oC on a horizontal rotator shaker for 1 hour.

Following incubation, cells were plated out at different concentrations onto 1.5% w/v LB/agar culture plates containing the appropriate selection antibiotic. Cell suspensions were allowed to absorb into the agar before the plates were placed in an incubator at 37oC for overnight culture.

Following overnight culture, individual colonies were picked using a pipette tip, and transferred to a sterile universal container containing 5 ml of LB medium

supplemented with selective antibiotic. This culture was again incubated overnight at 37oC in a rotatory shaker at 180 rpm. Plasmid DNA was isolated using the Miniprep technique described in 2.3.7.3 and analysed by restriction digestion.

2.3.7.3 Plasmid purification – Qiagen Miniprep

The QIAprep miniprep kit, Qiagen, was used for the isolation of small quantities of plasmid DNA for analysis or to take forward to further cloning steps. This kit uses a modified alkaline lysis protocol followed by binding of DNA to a silica membrane under low salt and pH conditions. Centrifuge wash steps remove endonucleases (buffer PB) and salts (buffer PE). DNA is then eluted in a small volume of buffer EB.

A culture of 5 ml LB medium with appropriate selection antibiotic was

inoculated with a scraping from a glycerol stock or with 100 μl from a previous culture. This pre-culture was incubated overnight in a rotatory shaker at 37oC. Two millilitres of culture was centrifuged at 8000 x g, 2 minutes and the

supernatant discarded. The pellet was resuspended in 250 μl buffer P1

supplemented with 100 μg/ml and Lyse Blue 0.1% v/v. Cell lysis was performed by addition of 250 μl buffer P2 followed by vigorous mixing and incubation at room temperature for 5 minutes. The lysis step was terminated by addition of 350 μl buffer N3 and immediate vigorous mixing. The resultant precipitate was centrifuged out at 18000 x g, 10 minutes. The supernatant was then transferred to a QIAprep spin column and centrifuged at 18000 x g, 1 minute. The flow- through was discarded. The spin column was then washed with 500 μl buffer PB followed 750 μl buffer PE, being centrifuged at 18000 x g, 1 minute for each wash. The spin column was centrifuged at 18000 x g, 1 minute to dry the membrane. DNA was eluted by addition of 30-50 μl buffer EB or nuclease free water to the membrane, incubation for 1 minute and subsequent centrifugation at 18000 x g, 1 minute. DNA concentration and purity were assessed by spectrophotometry using a Nanodrop 2000, following which the plasmid was stored at 4oC.

86

2.3.7.4 Plasmid purification – Qiagen Maxiprep

The Endofree Plasmid Maxiprep kit, Qiagen, was used for the large scale

purification of plasmids. This kit uses a modified alkaline lysis protocol, followed by removal of endotoxin and subsequent binding of supercoiled DNA to Qiagen Anion-Exchange Resin in the Qiagen-tip 500, under low salt and pH conditions. After wash steps, the purified DNA is eluted in a high salt buffer and then

precipitated using isopropanol.

A pre-culture of 5 ml LB medium with appropriate selection antibiotic was inoculated with a scraping from a glycerol stock or with 100 μl from a previous culture used for Plasmid Miniprep and analytical restriction digest. This pre- culture was incubated overnight in a rotatory shaker at 37oC. The following day, 4 ml of the pre-culture was inoculated into 400 ml of LB medium with

appropriate selection antibiotic and cultured overnight in a rotatory shaker at 37oC.

The bacterial culture was centrifuged at 6000 x g, 15 minutes, 4oC. The

bacterial pellet was resuspended in 10 ml buffer P1 supplemented with RNase 100 μg/ml and Lyse Blue 0.1% v/v to demonstrate homogeneous mixing. Cell lysis was performed by addition of 10 ml buffer P2 followed by vigorous mixing and incubation at room temperature for 5 minutes. The lysis step was

terminated by addition of 10 ml buffer P3 at 4oC and vigorous mixing. The resultant lysate was immediately poured into the QIAfilter cartridge and incubated at room temperature for 10 minutes to allow the precipitate,

containing proteins, genomic DNA and detergent to rise to the top. The lysate was then eluted from the cartridge into a fresh 50 ml container. Endotoxin was removed by addition of 2.5 ml buffer ER, mixing and incubation on ice for 30 minutes.

The Qiagen-tip 500 was equilibrated by applying 10 ml buffer QBT. The filtered lysate was then applied and allowed to pass through by gravity flow. The

Qiagen-tip 500 was washed by two additions of 30 ml buffer QC. Plasmid DNA was eluted with 15 ml buffer QN and precipitated by addition of 10.5 ml (0.7 volumes) of room-temperature isopropanol. The precipitated DNA was centrifuged at 15000 x g, 4oC, 30 minutes. The supernatant was carefully

decanted leaving the pellet intact. The pellet was washed with 5 ml 70% ethanol in endotoxin-free water and centrifuged 15000 x g, 4oC, 10 minutes. The supernatant was again carefully decanted leaving the pellet intact. The pellet was air dried and then dissolved in 100-200 μl buffer TE. DNA

concentration and purity were assessed by spectrophotometry using a Nanodrop 2000, following which the plasmid was stored at 4oC.

2.3.7.5 Plasmid analysis by restriction enzyme digestion and gel electrophoresis

Restriction enzyme digests were performed to validate the identity of isolated plasmids. Plasmid DNA was digested with a combination of enzymes chosen to provide fragments of known length, which could then be resolved by gel

electrophoresis. One microgram of plasmid DNA was digested with enzyme and the appropriate buffer (Fermentas) made up to 20 µl with sterilised deionised water. Digestion was performed at 37oC for 1 hour and terminated either by thermal denaturation or addition of 1 μl 0.5 M EDTA. Details of more complex digestions are provided in the individual methods sections.

Digestion products were resolved using TBE or TAE/agarose gels containing GelRed 0.01% v/v (VWR International), or stained using 2 ng/ml ethidium bromide. Gels were imaged using a GelDoc imager (BioRad).

2.3.7.6 Storage of plasmids as glycerol stocks

Bacterial cultures grown for plasmid isolation were frozen to allow initiation of future cultures. Aliquots of 700 μl of bacterial cultures were transferred to sterile 1.7 ml microtubes. Sterile glycerol, 300 μl, was added to each tube and mixed by inversion. These stocks were stored at -80oC.

88