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. Centrifuge cells at 4°C and 3000rpm for 10 min.

2. Decant media into a decon-filled beaker, leaving 0.5ml.

3. Transfer the pellet into an Eppendorf tube and spin at full speed (16,000rpm) for 30s; dispose of supernatant.

4. Resuspend with chilled 250μl Buffer P1 and transfer to a spin column.

5. Add in 250μl Buffer P2 (lysis buffer; if using Lyseblue reagent, solution will turn blue) and invert gently ONLY, for 4-6 times.

6. Add in 350μl Buffer N3 (neutralising buffer) and immediately mix the solutions, then invert gently for 4-6 times (if using Lyseblue reagent, milky mass will result with colorless solution containing plasmids).

7. Centrifuge for 10 min. at full speed.

8. Pipett the clear supernatant to spin column, being careful not to disturb the cell pellet, then centrifuge for 1 min. at full speed.

9. Empty the flow through, then add 750μl Buffer PE and centrifuge for 1 min. at full speed.

10. Empty the flow through and centrifuge again for 1 min. to dry the filtrate.

11. Transfer the spin column to an Eppendorf tube, then add in 50μl Buffer EB (10M Tris pH8, to elute DNA from the filter). Let stand for 2-5 min. to and then spin for 1 min. at full speed to elute DNA.

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.
 

5. 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:

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.