Team:TU-Eindhoven/LabJournal

Before any real lab work could begin some supplies had to be created, including a selection of agar plates. We needed two different types of agar plates, some with ampicillin antibiotics and others with kanamycin antibiotics. To manage this we created 2 separate agar solutions, a 200mL mixture for the ampicillin plates and a 400mL mixture for the kanamycin plates. The protocol we followed for the creation of the agar solutions was as follows:

• Mix together the following amounts to create 200mL agar solution:
  • 2g Peptones
  • 2g NaCl
  • 1g Yeast extract
  • 3g Agar
  • Fill the container up to 200mL with demineralised water.
• For a 400mL solution mix together:
  • 4g Peptones
  • 4g NaCl
  • 2g Yeast extract
  • 6g Agar
  • Fill the container up to 400mL with demineralised water.
• Next the containers were autoclaved and allowed to cool, but not harden.
• The following steps were all performed in the vicinity of a blue flame to ensure a sterile working environment.
• Before pouring the plates the correct antibiotics were added. The concentration of the ampicillin antibiotics was 100ηg/µL and that of the kanamycin antibiotics was 30ηg/µL.
• The solutions were now ready to be poured into agar plates. From the solutions we made we were able to pour 8 ampicillin plates and after receiving a little extra (about 350mL) kanamycin agar solution we poured a total of 27 kanamycin plates.
• The plates were cooled on the bench and allowed to harden before being stored in a 4° refrigerator.

Transforming the DNA

At this moment in time only one DNA vector (Protamine-1-Optimized) had arrived, but being impatient and rearing to go we decided to proceed with this one sample anyway which also allowed us a chance to get back into the rhythm of doing lab work. We needed to transform the 4µg of DNA into NB bacteria ready for plating and culturing. This all was done by completing the following steps:

• The first step was to dilute the 4µg of vector in 20µL of MilliQ water. This created a 200ηg/µL solution.
• Of the previously created solution 1µL was pipetted into 199µL of MilliQ water creating a 4000 times dilution (100ηg/µL).
• Of this 100ηg/µL solution 1µL was pipetted into 20µL of NB bacteria. The two dilutions could now be stored in the -20°C freezer.
• The NB/Vector solution was left on ice for a short while before being heat-shocked in a water-bath of 42°C for 30 seconds.
• Now the NB bacteria were returned to ice for 2 minutes.
• After allowing the bacteria to cool 80µL of SOC solution was added. Hereafter the NB bacteria were not returned to ice. Instead they were placed in a 37°C incubator for 60 minutes.

Plating the bacteria

After incubating the bacteria for a hour they were ready to be plated so that we could create a number of cultures. This was done in the following fashion:

• The following steps were all performed in the vicinity of a blue flame to increase the sterility.
• The plate was opened and the entire bacterial solution (approx. 101µL) was pipetted onto an ampicillin agar plate.
• To ensure even culture growth the solution was spread out over the plate using a sterile spreader.
• The plate with its bacterial spread was then placed in a 37°C incubator and left there overnight to grow.