Team:TU-Eindhoven/LabJournal

Creating agar plates

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 100ng/µL and that of the kanamycin antibiotics was 30ng/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°C 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 200ng/µL solution.
• Of the previously created solution 1µL was pipetted into 199µL of MilliQ water creating a 4000 times dilution (100ng/µL).
• Of this 100ng/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.

Making LB medium

Another preparational step that was performed was the creation of LB medium which we will be using a lot in the coming weeks. The protocol for the making of LB medium follows that for the creation of agar solution without the addition of the agar. So to create 1L of LB medium we mixed the following:

• 10g Peptones
• 10g NaCl
• 5g Yeast extract
• Fill the container up to 1L with demineralised water.
• The entire container was then placed in the autoclave and sterilized.
• The LB medium was allowed to cool and has been stored for later use at room temperature.

Creating small cultures

Today we continued with the Protamine-1-optimized sample. This sample had been plated onto an ampicillin agar plate and was allowed to grow overnight. The next step was to transfer cultures from this agar plate into small amounts of LB medium so we could obtain a slightly larger culture, essentially increasing the amount of viable DNA vectors we have. The transformation to LB went as follows:

• All following steps were performed in the vicinity of a blue flame increasing the sterility.
• Firstly three small falcon tubes were filled with 8mL of LB medium.
• To each of the falcon tubes 8µL of ampicillin antibiotics were added.
• Now the agar plate was opened and three free lying colonies were chosen for picking. Each of the chosen colonies was then picked by lightly scraping across it with a pipet point. Each pipet point and colony picking was then ejected into one of the falcon tubes.
• The falcon tubes were then placed into a rotating incubator set to 37°C and left there to culture overnight.

As there were no further preparational steps to perform and no other DNA vectors had arrived this concluded a rather short day in the lab.

DNA Retention from cultures