1. Let frozen cell thaw from -80 °C and warm only with finger tips when using.

2. Mix 10 μL of cells with 1 μL of vectors, and leave on ice for 30 min.

3. Put 1 mL of LB liquid medium into Eppendorf tube, warm up briefly in 37°C.

4. Put the cell and vector mixture on 42 °C water bath for 45 secs. to heat shock the cells, allowing membrane holes to close.

5. Pipette 50 μL of warm LB liquid medium into the tube with mixture.

6. Shake at 37 °C for 30 min; recombinant E.coli will start to grow.

7. Spread all contents of the shaken tube onto agar plate and incubate at 37 °C overnight.

8. Pipette using 10 mL pipette and pipette gun to transfer 10-15 mL (leave enough air for bacterial growth) of liquid LB medium in four 50 mL Falcon tubes; one labelled tube per colony.

9. Harvest recombinant bacteria from agar plate by scratching gently with the scraper. The plate should be facing downwards to minimise contamination.

10. Put the scraper tip into the Falcon tubes to transfer the bacteria into medium.

11. Add into the Falcon tubes 1 μL of ampicillin (1 μL for 10 μL).

12. Incubate the bacteria on shaker incubator at 37 °C overnight but not excess 20 hr (otherwise the plasmids will be expelled from the cells).

1. Preparing 1% agarose solution: For 100 mL, dissolve 1 g agarose powder in 100 mL TAE (Tris base, acetic acid and EDTA) and heat in microwave. Then, add ethidium bromide (8 μL for 100 mL).

2. Pour solution into plastic holder, add comb, and allow to cool - gel will set.

3. Load samples into wells (5-20 μL), also adding DNA ladder.

4. Run at 80 V for 20 min. to 50 min. depending on size of gel.

5. Visualize under UV light.
 

6. 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. Prepare the sample in the following sequence (30μl):

2. Incubate in 37 °C water bath for 1 - 12 h, depending on brand of restriction enzyme.

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. Prepare multiple PCR tubes for each plate and make a solution as follows:

to make a 50μL PCR solution

2. Prepare and label corresponding Eppendorf tubes for each PCR tube

3. Using a small pipette tip, scratch one colony off a plate and dip the tip into the PCR tube. Store the tip in the corresponding Eppendorf tubes (for further incubation, if positive).

4. Run PCR using optimal annealing temperature for the required gene, and then take samples of the PCR products for electrophoresis.

6. Select the positive from all samples and incubate the corresponding tips.

Ligation molar ratio calculation:

Compare UV light exposure intensity for quantity 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. Prepare 20 μL of ligation solution

• 2 μL 10x buffer
• 1 μL T4 ligase
• ~17 μL consist of vectors and insert, according to calculated ratio
• make up to 20 μL using water

2. Let stand at room temperature for 10-30 min.

3. Mix the ligase-treated vector-insert mixture with 60uμl E. Coli (DH5-α), then proceed the standard transformation protocol and shake for 1 hr; finally add 200 μL LB liquid media.

4. Spread all contents of the shaken mixture onto agar plates, with an additional control of transformed E. Coli with non-inserted linearised vector. Incubate overnight at 37 °C.

1. Using LB as a blank reference, measure the optimal density of 1 mL of sample in the photometer.

2. For induction, the OD should be at 0.5. The sample can be grown further or diluted if necessary.

2. IPTG should be added to a concentration of 1 mM. This figure will vary if protein expression/cell growth is carried out at a lower non-standard temperatures to accomodate for the slower metabolism.

1. Spin down the bacteria in a centrifuge.

2. Discard the supernatant and add in 1 mL of PBS (phosphate buffer saline) to resuspend the pallet, then tansfer to Eppendorf tubes.

3. Spin down the bacteria at full speed for 1 min., discard supernatant and then put on ice/in cool room for storage.

4. Prepare lysozyme solution (10 mg/mL) in lysis buffer.

5. Add 200 μL into each pallet and resuspend it; vortex and shaker incubate at 37 °C for 30 min. to 1 h.

6. Sonicate the cells.

7. Take 10 μL total sample and add in 10 μL 2x dye.

8. Spin the original tubes for 1 min. at full speed, remove the supernatant, then resuspend the mass with 200 μL PBS.

9. Take 10 μL membrane sample from the resuspended solution and add in 10 μL 2x dye.

10. Denature the protein samples on the heating block at 95° C or 75°C (for membrane protein).

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

1. Spin down the bacteria in a centrifuge.

2. Discard the supernatant and add in 1ml of PBS to resuspend the pallet, then transfer to Eppendorf tubes.

3. Spin down the bacteria again at full speed for 1 min. and discard supernatant.

4. Resuspend the pallet with 100 μL PBS and then transfer to glass tubes.

5. Add organic solution for lysis: 375μl of chloroform:methanol 1:2.

6. Vortex in the cold room for 1h and then add 125μl chloroform and 125μl water to allow tpahse serparation (lower organic layer contains lipids); let stand for at least 10 min.

7. Spin down using special adapters at 3000rpm for 5 min.

8. Pipette the bottom layer into a glass sample tube, being careful not to take any milky layer.

9. Dry the organic solvent under nitrogen and store.

Primer annealing was carried out to create short peptides.

10 μL of 200 μM of each primer was put into the PCR machine.
 

PCR Programme: