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 (1ng/µL).
• Of this 1ng/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.

Transforming DNA

Today a further 4 constructs arrived (Poly(Arginine-Serine), Poly(Arginine-Glycine), 1PJN1 and 1ETF) so it became possible to continue with the lab work: increasing the amount of viable DNA for these samples much like we had done for the Protamine-1-optimized sample which we had recieved a week earlier. To increase the DNA we would transfer the DNA into Nova-Blue bacteria and culture these allowing the bacteria to replicate our own vector constructs. To isolate the DNA we would then move on to miniprepping the cultures. Firstly however the would need to be transformed into the bacteria. The steps taken to do this are listed below:

• Before transformation could be started the 4µg of DNA we had recieved would need to be diluted to a 1ng/µL concentration as follows:
  • 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 (1ng/µL).
• With this 1ng/µL solution the transformation could commense:
  • Of this 1ng/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.

Creating the Cultures

Looking ahead to the cloning we would like to perform later on and using the yields of DNA obtained from the Protamine-1-Optimized sample last week a small calculation was performed to see how much DNA we would need and therefore how many cultures would need to be created. As we were going to attempt to use gel extraction to purify the digestion products of these constructs we would need to culture approximately 20 times more DNA than that we would eventually need for the ligation. Based on the average 150ng/µL DNA yield from the Protamine-1-Optimized construct we determined we would need 12 8mL cultures per construct to obtain the correct amount of DNA. This meant that we would would be creating:

• 12 cultures of Poly(Arginine-Glycine).
• 12 cultures of Poly(Arginine-Serine).
• 12 cultures of 1PJN1.
• 12 cultures of 1ETF.
• 9 cultures of Protamine-1-Optimized to bring the total number of cultures up to 12.

Again looking ahead to the cloning we saw that we would also need to increase the amount of pBR322 and pET28a vectors we had. For each of these we would need to create 3 8mL cultures to be able to perform an equimolar ligation. This all brought the total number of cultures we would need to create up to 63. The creating of the cultures was then performed in the following manner:

• All the following steps were performed in the vicinity of a blue flame to increase the sterility.
• 63 culture tubes were filled with 8mL of LB medium which had been prepared earlier.
• 8µL of 1000x Ampicillin antibiotics were then added to 60 of the culture tubes. 8µL of 1000x stock Kanamycin antibiotics were then added to the remaining three culture tubes. This was done as the pET28a vectors were resilient to Kanamycin and not to Ampicillin which all the other constructs were.
• The culture plates for each of the constructs were then retrieved. Instead of, as we did with the previous Protamine-1-Optimized constructs, pipetting a single culture into each of the culture tubes we now to decided to pick a culture and dissolve it in 15µL of water. From this 15µL solution 5µL could be pipetted into a culture tube. This meant that we would not have to pick 12 cultures from the plate per construct (or 9 for the Protamine-1-Optimized construct) but only 4 (or 3 for the Protamine-1-Optimized construct).
• For the pET28a culture, an eppendorf tube containing a glycerol stock of pET28a vectors in NB bacteria was retrieved and from the -80°C freezer and once thawed, 5µL was pipetted into each of the 3 culture tubes.
• For the pBR322 culture we would first have to transform the DNA into NB bacteria in the following fashion:
  • Firstly the pBR322 vector DNA would have to be diluted as follows:
    • Add 450µL of MilliQ water to the DNA to create a 100ng/µL stock solution.
    • Take 1µL of the 100ng/µL stock solution and add it to a further 99µL of MilliQ water creating a 1ng/µL stock solution.
  • Using this 1ng/µL stock solution the transformation could commense.
    • 1µL of the stock solution was added to 20µL of NB bacteria.
    • This solution was left on ice for 5 minutes before being placed in a 42°C water bath, heat shocking the bacteria.
    • The bacterial solution was then placed on ice for a further 2 minutes before 80µL of SOC was added to the solution.
    • Without placing on ice first the solution was then placed in a 37°C incubator for 60 minutes.
  • After 60 minutes in the incubator the 20µL of the solution could be pipetted into each of the three pBR322 culture tubes.
  • 60µL of SOC was subsequently added to the remainder of the bacterial solution so that it could be plated (on an Ampicillin agar plate) and in that way serve as a backup should the culture not grow.
• Now that all the culture tubes were complete they could be placed in a 37°C incubator overnight to grow.