Team:TU-Eindhoven/LabJournalTest

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

Previously (on the 25th July 2013) three small 8mL cultures had been placed in the incubator containing NB bacteria which housed the vector for the protamine-1-optimized sample. These cultures had grown overnight and it was our aim today to retain the DNA vectors from these three cultures, essentially having allowed us to multiple the amount of DNA we had available. To perform this DNA retention a miniprep protocol was followed as is described below:

• First the culture tubes were spun down in a centrifuge for 10 minutes at 3700rpm causing the bacterial cells with the DNA inside to form pellets isolating it from the growth medium.
• The supernatant that had formed above the pellets was discarded and the pellets themselves were resuspended in 250µL of P1 buffer. The tubes were gently shaken by hand until the entire pellet had resuspended.
• The suspension was then transferred by means of pipetting into a smaller (1.5mL) eppendorf tube.
• 250µL of P2 buffer was then added to the eppendorf tube and the solution was inverted by hand until it had turned a clear blue.
• Within 5 minutes of adding the P2 buffer 350µL of N3 buffer was added to the solution and the eppendorf tube was inverted again until the solution was clear again.
• Once clear the eppendorf tube was placed in the centrifuge and spun for 10 minutes at 13000rpm seperating the bacterial cells from the DNA which remained suspended in the solution. The bacterial cells formed a pellet in the bottom of the eppendorf tube. (Unfortunately the pellet had not completely formed after 10 minutes so the eppendorf tube was spun for a further minute, again at 13000rpm.)
• As soon as the centrifuge had stopped and we could see that the pellet had properly formed, the supernatant was poured off into a special QIAcolumn. The pellet could be discarded in the bio-hazard waste.
• The QIAcolumn was subsequently centrifuged for one minute at 13000rpm, during which the DNA vector bound to the column meaning the flow through could be discarded.
• 750µL of PE buffer was then added to the QIAcolumn before centrifuging it for another minute at 13000rpm. The flow through was discarded.
• To ensure that all the PE buffer had passed through the column it was then spun yet again for one minute at a speed of 13000rpm. Yet again the flow through could be discarded.
• The column itself was then placed above a sealable 1.5mL eppendorf tube so that the DNA could be contained and stored. To obtain the DNA from the column 50µL of MilliQ water was pipetted onto the very center membrane of the column after which it was spun down for one minute at 13000rpm. The flow through now sat in the 1.5mL eppendorf tube and contained the DNA vectors.

Nanodrop Test

To check how much DNA we had acquired by culturing the NB bacteria with our vector a small nanodrop test was performed. This would tell us how many ng of DNA could be found in each µL of our samples. The results have been given below:

• Tube 1 contained 145.5 ng/µL of DNA.
• Tube 2 contained 164.1 ng/µL of DNA.
• Tube 3 contained 153.9 ng/µL of DNA.

The remaining DNA was then placed in a -20°C freezer and stored for later use concluding the days lab work.