1. Frozen cells were thawed from -80 °C.

2. Thawed cells (10 μL) were mixed with vector (1 μL), and left on ice for 30 min.

3. LB liquid medium (1 mL) was warmed at 37°C for 30 min.

4. The cell/vector mix was heated at 42 °C for 45 seconds.

5. Warm LB (50 μL) was added to the cell vector mix.

6. Mixture was shaken at 37 °C for 30 min.

7. Mixture was plated onto ampicillin agar plates and left overnight.

8. Recombinant E. Coli was harvested from agar plate by scratching gently with the scraper.

9. The scraper tip was placed into Falcon tubes with LB (10 mL) and ampicillin (1 μL) to transfer the E. Coli into medium.

10. The E. Coli was incubated in a shaker at 37 °C for less than 20 hr.

1. Preparing 1% agarose solution: Agarose powder (1 g) was dissolved in TAE (Tris base, acetic acid and EDTA) buffer (100 mL). This was heated in the microwave until the solution was transparent. Ethidium Bromide (8 μL) was added.

2. The solution was poured into an assembled gel rigand allowed to set.

3. Prepared samples were loaded into the wells(5-20 μL) along with DNA ladder (5 μL).

4. The gel was run at 80 V for 20-50 min.

5. The gel was visualised under UV light
 

Ratios for sample preparation vary depending on the type of sample:

• Miniprep - use 4 μL sample and 2 μL dye
• Digestion - use 1 μL sample and 2 μL dye
• PCR- use 10μl sample and 1μl dye

When using GoTaq®, simply load 10 μL onto the gel, as dye is already included.

1. The sample (30μl) was prepared in the following sequnce:

2. The sample was incubated at 37 °C in a water bath. Timings depended on the restriction enzymes brand.

1. Prepare 20 μM primer working stock for both forward and reverse primers

2. Prepare PCR according to polymerase brand:

We used Epoch BioLab's GenCatch PCR Cleanup Kit (Protocol) and Qiagen's QIAquick Gel Extraction Kit (Protocol).

1. PCR tubes were prepared in the following sequence:

to make a 50μL PCR solution

2. An Eppendorf tube was prepared for each PRC tube.

3. Using a small pipette tip, one colony was scratched off a plate and dipped into the tip into the PCR tube. Tips were then stored in the corresponding Eppendorf tubes (for further incubation, if positive).

4. The PCR was ran using optimal annealing temperatures for the required gene, and then the samples of the PCR run straight on electrophoresis.

6. Positive results from the samples were incubated from the corresponding tips.

1. Add phosphatase buffer to sample in accordance with its concentration factor.

2. Add 1μL phosphatase.

3. Incubate 60 mins at 37°C.

4. Heat-shock for 5 mins at 65°C.

Ligation molar ratio calculation:

Exposure intensity from UV light was used to quantify the DNA in ng (i.e. the concentration of both insert and vector) against ladder position for sequence length (i.e. molar weight ratio of the two)
Rule of 3: Three times as much insert as vector ensures successful ligation results

1. Ligation solution (20 μL) was prepared

• 10x buffer (2 μL)
• T4 ligase (2 μL)
• Vectors and insert (~17 μL), according to calculated ratio
• make up to a total of 20 μL using water

2. The ligation mixture was allowed to stand at room temperature for 10-30 minutes..

3. The ligase-treated vector-insert mixture was mixed with E. Coli (DH5-α)(60 μL), then the standard transformation protocol was followed with shaking for 1 hr; finally LB (200 μL) was added.

1. The optimal density (OD) of a portion of the sample (1 mL) was measured in the spectrophotometer using LB as a blank.

2. If the OD was approximately 0.5 the sample was induced. The sample could be incubated further or diluted if necessary.

2. IPTG (1 mM) was added.

1. The E. Coli was spun down in a centrifuge.

2. The supernatant was discarded and phosphate buffer saline(PBS, 1 mL) was added to resuspend the pellet before the mix was transferred to an Eppendorf tube.

3. The E. Coli was spun at full speed for 1 min., the supernatant discarded and the pellet was put on ice for storage.

4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.

5. Lysozyme solution (200 μL) was added to each pellet and they were resuspended and vortexed before being incubated in a shaker (37 °C) for 30 min. to 1 h.

6. The cells were sonicated.

7. Total sample (10 μL) was removed and 2x dye (10 μL) was added.

8. \the original tubes were spun for 1 min. at full speed and the supernatant was removed, the mass was resuspended with PBS (200 μL).

9. Membrane sample (10 μL) was taken from the resuspended solution and 2x dye (10 μL) was added.

10. The protein samples were denatures on a heating block (95° C or 75°C for membrane protein).

11. The samples were cooled on ice for 1 min., then loaded on to the protein gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for around 45 min.

1. Making the membrane transfer sandwiches: 2x A2 at the bottom, then 2x A2.

2. The membrane, GE Healthcare HyBond ECL films, was cut out using a glass template; the membrane was put onto moist A2 filter papers and pressed flat using a roll.

3. The gel slides were opened using a wedge; removed the thinner slide and, using the wedge, prized the protein gel from the thicker plate and onto the membrane.

4. The airbubbles were gently pressed out between the membrane and the gel.

5. CAT filter paper was added.

6. The power pack was run for 1hr (40 mA).

7. If the restained markers have transferred, place the CAT filter papers onto a rocker and add stain. If using Ponceau, the stain can be reused.

8. Destain was done by rinsing with water.

21. Milk powder (50 mL 5%) was added and left to rock for 2 hours at room temperature. If leaving overnight, rock in cold room using lower speed.

9. The blocking milk solution was decanted, and replaced with milk (10 mL 1%) with antibodies (5μl of diluted). This was rocked for 1 h.

10. The milk-antibody solution was poured off and washed with PBS (0.3%) and Tween, three times at 10 min intervals.

11. A solution of "Supersignal west Fermto trail" kit (1:1 ratio, 1 mL per membrane).

12. The membrane was made moist with the visualization solution, covered for 5 min, then the excess liquid was removed.

13. The membranes were developed in a dark room.

1. The E. Coli was spun down in a centrifuge(800g, 10min).

2. With the supernatant discarded, PBS (1 mL) was used to resuspend the pellet, then transferred to Eppendorf tubes.

3. The E. Coli was spun again at full speed for 1 min. and the supernatant discarded.

4. The pellet was resuspended using PBS (100 μL) and then transferred to glass sample vials.

5. An organic mixture of chloroform:methanol (1:2(v/v), 375 μL) was added for lysis.

6. The sample was vortexed for 1 h in the cold room, with chloroform (125 μL) and water (125 μL) added to allow phase separation (lower organic layer contains lipids); and left to stand for at least 10 min.

7. The sample vials were spun at 1000g at room temperature for 5 min.

8. The lower organic layer was transfered to a new glass vial avoiding the upper aqueous layer at all costs.

9. The resultant lower phase lipid extract was then dried under N₂, and the fatty acid stored in the fridge.

9. The samples can be re-dissolved in chloroform:methanol (1:2) and are ready for Electrospray-mass spectrometry analysis for phospholipids and can be further processed for fatty acid derivation and analysis by Gas Chromatography-Mass Spectrometry.