Our Protocols

Fill a container with 400ml distilled water
Add 25 g/L of LB broth to the distilled water
Swirl the container with the water and LB broth together so that the powder has nearly dissolved
Autoclave the container and store in fridge at 4 °C until LB broth is required

Fill a container with 200 ml distilled water
Add 25 g/L of LB broth and 10 g/L Agar to the container
Swirl the container so that both the LB broth and Agar are nearly dissolved and autoclave
Add 200 µl of antibiotic (Amp, Chl, Kan etc.) to the autoclaved container along with 100 µl X-gal and 100 µl IPTG
Distributed finalised mixture equally between 8 petri dishes, making sure all the plates are completely covered and leave to set
Once set, store the plates at 4 °C until required.

To a 1.5 ml Eppendorf tube, add 50 µl of competent cells to 10ul of DNA and leave on ice for 10 minutes. Heat shock at 42 °C for 1 min before putting the cells back on ice for 5 min. Following this, add 1 ml LB broth with no antibiotic before shaking in the incubator 1 hour at 37 °C. On a chloramphenicol, X-gal and IPTG plate, add 5-10 glass beads with 200 ul of cells and DNA from incubation. Shake the plates until the cells and DNA are equally distributed across the plate. Remove the beads and incubate the plate at 37°C overnight.
If the colour of the colonies is indecipherable after incubation, place in 4 °C fridge until colour develops.

From plate showing successfully transformed white colonies, pick the appropriate colonies using a pipette tip and place into 5ml LB broth with suitable antibiotic (Amp, Chl, Kan etc.) Resuspend the colony in the LB broth and antibiotic before vortexing for 10 seconds. Incubate tubes at 37°C in the shaking machine overnight.

Place Buffer EB (for step 10) into 55 °C water bath. Pellet 1-5ml bacterial overnight culture by centrifugation at 13,000 rpm for 3 min at room temperature (15-25 °C).
Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
Centrifuge for 10 min at 13000 rpm in a table-top microcentrifuge.
Apply 800 µl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60s and discard the flow-through.
Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60s and discard the flow-through
Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60s and discard the flow-through.

Team:Cardiff Wales/protocols