Week One: 1/22-1/26 Growth and Extraction of the pRHAM Plasmid

 Monday

On Monday, I started out the week by preparing LB Broth and LB Broth with Agar. I made LB media because the pRHAM plasmid is extracted from E.Coli which can only be grown on LB. So, I made 200 mL of LB Agar to prepare around 8 plates once autoclaved and heated, but that will be touched on in a later day. Along with the LB Agar I had also prepared 250 mL of LB Broth which will be used to make smaller flasks of LB to grow the E.Coli in. Once both of those broths were prepared I put them on the autoclave tray and waited for them to be autoclaved for use the next day. Before I left for the day however, I calculated how much kanamycin I needed for both the broth and the broth with agar. I did this by getting the stock concentration that we had, which was 7.5 mg/mL, finding the desired concentration which was 30 ug/mL and then using the desired volume which was either 200 mL for the agar or 250 mL for the broth without agar. The reason kanamycin was used is because that specific E.Coli with the pRHAM plasmid had a kanamycin resistant gene that we wanted, so if we didn’t use the kanamycin then we would have other types of E.Coli that we don’t want.

Tuesday

On Tuesday, we prepared 4 25 mL flasks of the autoclaved LB Broth so that we could inoculate the flasks with the E.Coli and get growth for plasmid extraction. While doing this we heated up the 200 mL of LB Agar and got 8 plates ready to be poured once the agar had melted. Once the agar had melted we let it cool enough so that we could handle it and that it wouldn’t burn out the kanamycin. Before pouring the plates we put in 800 uL of the kanamycin as that was how much I calculated from the day before to get the desired concentration of kanamycin. We were hopeful that we could inoculate the plates with the pRHAM E. Coli that day if they cooled down fast enough but sadly that wasn’t the case so we had to wait until the next day to inoculate. We spent the rest of our day coming up with a possible schedule for the rest of that week and some part of the coming up week, however it was subject to change due to the growth of the E. Coli and success with the plasmid extraction later that week. 

Wednesday

By now our plates were solid enough for us to inoculate so we started up the hood and followed the procedures posted on it to make sure everything was sterile because we had to pull the E.Coli from freezebacs so we didn’t want to any possibility of contamination anywhere. While working under the hood we did the 4 way streak method to get as much growth as we could on our plates. We only streaked 4 plates with the E.Coli and left the other 4 plates in the fridge if we needed to grow more E.Coli. Once we had streaked our plates and were done with the hood we wiped everything down with ethanol, turned off the blower, closed the hood lid, and turned on the UV light for 15 minutes. Next, we took our plates to the the 37 C incubator in the back room since E. Coli grows best at 37 C. So for the next day if growth had occurred we could inoculate from plate to broth so that we can do the plasmid extraction by Friday, and we were going to gram stain the E.Coli to make sure that there were no contaminations before inoculating. 

Thursday

By Thursday we had gotten the growth that we were looking for with the E. Coli in the plates, now we had to gram stain the plates to make sure that there wasn’t any contamination and then inoculate from plate to broth. We had done two separate plates to get more reliable results from the stains. On our first stain we had gotten purple dots all throughout the cultures that we had picked up, I thought that it was staph contamination but looking at pictures of staph in gram staining the circles were too big and not clumped next to each other, so it was unlikely that it was staph contamination. So we decided to do a second gram stain but change the way we go about the ethanol step. Usually we would leave the ethanol on the slide for about 15 seconds to get rid of the crystal violet, however we learned that if we drip the ethanol on to the slide and let it drip off, and repeat a couple times that it gets ride of the crystal violet stain more effectively. By utilizing this change in procedure we looked at our 2nd gram stain, and it worked. My slide had no more purple dots all over and my E. Coli was the pink color that it should be. Thankfully the dots were just excess crystal violet stain and not contamination. With this successful gram stain we were not able to inoculate 2 of the 4 flasks that we prepared on Tuesday. With those now inoculated we put them into the shaking incubator and let them incubate for nearly 24 hours. 

E. Coli Gram Stain with Purple Dots 
E. Coli Gram Stain with Purple Dots

E. Coli Gram Stain After Ethanol Wash


Friday

  When we came into the lab that day we were ready to extract the pRHAM plasmid using the Zyppy Plasmid Miniprep Kit. We decided to do 4 extractions to make sure that we got reliable results. We followed the protocol on the kit exactly, the only difference is that we packed the cells before we started by adding 600 uL of the culture to a micro-centrifuge tube and put it in the centrifuge for about 30 seconds. We did this to get the bacteria packed together and more likely to react with the protocol. By the end we got 4 pRHAM plasmids ready for PCR the next week. 

Overall the week was mostly preparation getting ready for the plasmid extraction and then PCR for the next week so that we can get many copies of the plasmid. With the PCR product of this plasmid we would have everything we needed to start Gibson Assembly. We have the pRHAM plasmid, the pRAD1 plasmid, and D.Rad DNA /competent cells. 


Lorenz Christian Reimer, Joaquim Sardà Carbasse, Julia Koblitz, Christian Ebeling, Adam Podstawka, Jörg Overmann, BacDive in 2022: the knowledge base for standardized bacterial and archaeal data, Nucleic Acids Research, Volume 50, Issue D1, 7 January 2022, Pages D741–D746, https://doi.org/10.1093/nar/gkab961

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