Sp'23: Week 10 (3/27/23 - 3/31/23): Ventures with ZymoPURE

This week, I focused on attempting to extract pRad1 from E. coli using the ZymoPURE Plasmid Miniprep Kit [Box A] and the four LB + Ampicillin [50 ug/mL] tubes that I had inoculated into last week (tubes "3.24B1" - "3.24B4") using Jonathan's plate of E.coli + pRad1: 

Unfortunately, the plasmid extraction quantities of each of these tubes was less than ideal. Their respective dsDNA readings using the nanodrop show low plasmid yield (of less than 30 ng/uL each), with each tube's respective plasmid quantity listed: 


Now, a dsDNA reading of at least 100 ng/uL is desired after plasmid extractions are performed, as a higher yield would likely result in a higher chance at transformation due to a higher presence of DNA meaning a stronger chance at D. aquaticus's DNA uptake in transformation. Because of this (and what I should have done initially), I checked the growth of the original tubes that had been used for extraction (tubes "3.24B1" - "3.24B4"), and the growth had numbers that would have made a decent extraction possible, but the OD600 numbers showed growth that was less than ideal nonetheless (an A600 number of at least 1.00 is desired): 

After plasmid extraction had been attempted on all four tubes, they were placed back into the incubator for a second attempt at extraction two days later. However, before extractions were done again, I decided to do a gram stain on each of the four E. coli with pRad1 tubes. I'm quite glad I did, as three of the four tubes (tube "3.24B2," "3.24B3," and "3.24B4") showed contamination, while tube "3.24B1" was the only plate that did not seem to be affected by contamination: 

 

 

Because of this, I went forward with a plasmid extraction on only "Tube 3.24B1" using the ZymoPURE Plasmid Miniprep Kit [Box B] once more, which still resulted in a low plasmid yield in the end (a dsDNA reading of 14.7 ng/uL): 


Because of this, I decided that I needed to re-inoculate E. coli with pRad1 altogether and attempt to try for some fresh growth. Because of this, I created 200mL of LB that could be used for inoculations next week.

Comments

Post a Comment

Popular posts from this blog

Week 11 (11/14/22 – 11/18/22): Inventory & Prep

Image
This week's focus was on preparing for getting what I can done within the next 4 weeks before it is time to wrap up the semester for the holiday break. One thing I did implement was creating a more streamlined process of keeping track of our inventory so that we can always know how much of what material (and of what bacteria) we have in stock at all times. Because of this, I created quite a bit of broth, both with and without agar, so we can have what we need for the next few weeks. The following was created:  - 200 mL of LB broth (will be used for any kind of inoculation of E. coli)  - 200 mL of TGY 3:3:1 broth (will be used for any kind of inoculation of Deinococcus radiodurans or Deinococcus aquatics)  - 200 mL of LB broth with agar (will have ampicillin added to it when creating plates and will be used to inoculate any of our E coli that have plasmids with the Ampicillin-resistant gene)  - 75 mL of LB broth with agar (will be used to inoculate regular E coli onto...
Read more

Week 3 (9/19/22 – 9/23/22): Back to the Drawing Board

Image
  This was the week where after running transformation and plating on Friday (a third attempt), we were hoping that we would have transformed Deinococcus radiodurans such that it would have shown antibiotic resistance via integration of the pRADZ3 gene into its own genome. However, this unfortunately didn’t happen, for reasons that we are unsure of (so far). When we checked our plates on Day #4, 5, there was still no growth, indicating no transformation. Because of this, it is time to go back to the drawing board and are looking over every piece of the transformation protocol, from inoculation of E. coli (with its necessary plasmid that will be extracted for transformation) and   inoculation of D. rad (which is the species of Deinococcus that will be used for transformation) to plating of the hopefully transformed bacteria.   Now, an interesting factor to consider while looking at steps to transformation [on paper] for a third time   is to consider a factor that ...
Read more

Week 13 (12/5/22 – 12/9/22): Preparation for One More Trial

Image
This week, since we only have one week left of school until the semester ends, we are in the wrap up phase to where we have discovered that we can do one more transformation run. This will be our last chance of the year to see if we can attempt transformation without contaminating any of our items, as this seems to me the most prominent issue that we have had throughout the semester. Now, what was interesting was that we decided to do gram stains of all of the bacteria that we used for a transformation that was immediately taken out of the shaking incubator on day two of transformation, right before we plated them. We actually thought that we would find contamination in at least one of these tubes, but alas, every single one of them was a perfect gramstain of just the bacteria we needed. Because of this, and because the gram stains of all of our competent cells from freeze back where sterile, we could only then conclude that contamination occurred on transformat...
Read more