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

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

3. Put 1ml 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 45s to heat shock the cells, allowing membrane holes to close.

5. Pipette 50μl 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 10ml pipette and pipette gun to transfer 10-15ml (leave enough air for bacterial growth) of liquid LB medium in four 50ml 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 1ul of ampicillin (1μl for 10μl).

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

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

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

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

4. Run at 80V 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 4μl sample and 2μl dye
• Digestion - use 1μl sample and 2μl dye
• PCR- use 10μl sample and 1μl dye

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

1. Prepare 20μM 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: 23μl water 25uμl GoTaq MasterMix buffer 1μl of each primers 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 20μl of ligation solution

• 2μl 10X buffer
• 1μl T4 ligase
• ~17μl consist of vectors and insert, according to calculated ratio
• if needed, make up to 20μl 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-alpha), then proceed the standard transformation protocol (but shake for 1hr); finally add in 200μl 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.