Protocols

Chemically Competent E. coli Cells

1. Dilute overnight culture of E. coli cells 1/100 into 5 ml of fresh LB.
2. Incubate cells at 37°C with shaking until an OD600 of 0.5-0.8 is reached.
3. Chill the cells on ice for 20 min before harvesting at 4000 rpm at 4°C for 10 min.
4. Resuspend pellet in 0.5 ml ice cold transformation buffer (TB) and leave on ice for 10 min.
5. Divide cells into 100 µl aliquots, snap freeze in liquid nitrogen and store at -80oC.

Transformation of chemically competent E. coli cells

1. Thaw 100 µl of competent cells on ice for 20 min and mix with either 1 µl of plasmid DNA or 10 µl ligation reaction.
2. Chill cells on ice for 20 min.
3. Heat shock cells at 42°C for 90 seconds, before chilling cells on ice for 2 min.
4. Add 1 ml of LB to cells and recover at 37°C with shaking for 1 hour.
5. Pellet cells by centrifugation at 13 000 rpm for 3 min.
6. Remove supernatant resuspend in 200 µl of LB and plate onto LB medium containing appropriate antibiotics.
7. Incubate plates overnight at 37oC.

Polymerase Chain Reaction

1. PCR reaction mix set up as follows (final volume = 50 µl)

2. Thermocycler protocol (Elongation time was altered according to desired length of PCR product).

3. Run products on a 1% agarose gel for analysis.

Agarose Gel Electrophoresis

DNA products are run on a 1% (w/v) agarose gel to separate charged nucleic acids by size for analysis or purification. GelRed used to stain nucleic acids and gels visualised on a BioRad GelDoc.

For 1% (w/v) agarose gel

1. Weigh out 1g of agarose and pour into 100 ml of 1 x tris-acetate-EDTA (TAE) buffer.
2. Heat agarose solution in the microwave until the agarose had dissolved (1-3minutes) and there is a rolling boil.
3. All the solution to cool before Gel Red (6µl per 100ml of 1% (w/v) agarose solution) is added.
4. Pour agarose solution into a gel tray with the required combs in place.
5. Allow the agarose solution to cool in the gel tray before loading the DNA into the gel.
6. Mix the DNA samples with 10 x DNA loading dye and load on the gel with the DNA marker (Roche).
7. Run the gels in 1 x TAE buffer for 30 min at 100 V. After sufficient separation the gel is visualised under UV light on the BioRad GelDoc.

TAE buffer recipe (pH 7.6): 40mM Tris, 20mM acetic acid, 1mM EDTA

QIAquick Gel Extraction Kit protocol (Qiagen)

QIAquick Gel Extraction Kit Protocol from Qiagen was used. This was designed to extract and purify DNA from agarose gels in TAE buffer.

1. Excise DNA from agarose gel with a clean sharp scalpel and add into a tube.
2. Add 800 µl of buffer QG of gel slice.
3. Incubate at 50°C for 10 min or until gel slice has completely dissolved. To help dissolve gel, mix by vortexing the tube every 2-3 minutes.
4. After gel slice has dissolved completely the colour of the mixture should be yellow.
5. Add 200 µl of isopropanol to sample and mix.
6. Add 500µl of buffer QG to the QIAquick column and centrifuge for 1 minute. Discard flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 µl buffer PE to QIAquick column and centrifuge for 1 minute. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the QIAquick column in the 2ml collection tube provided for 1 minute to remove residual wash buffer.
8. Place the QIAquick into a clean 1.5 ml microcentrifuge tube
9. To elute DNA, add 50 µl of water to the centre of the QIAquick membrane and centrifuge for 1 minute.
10. To analyse purified DNA of a gel, add 1 volume of loading dye to 5 volumes of purified DNA.

Chemically Competent E. coli Cells

1. 1. Thaw 100 µl of competent cells on ice for 20 min and mix with either 1 µl of plasmid DNA or 10 µl ligation reaction.
2. 2. Chill cells on ice for 20 min.

Chemically Competent E. coli Cells

1. 1. Thaw 100 µl of competent cells on ice for 20 min and mix with either 1 µl of plasmid DNA or 10 µl ligation reaction.
2. 2. Chill cells on ice for 20 min.