Week 9 (10/31/22 – 11/4/22): Gram Stains & Gels

I made an interesting discovery this week which actually involved a process that I should have suspected but didn't think was happening at the time. Last week, when I did my Gram stain on my bacteria, I thought that because I saw pinky coli in the microscope after having done the gram stain, with regions of purple, that I had just done the Gram stain and correctly as otherwise the second and third slides came out just fine. However, my lab mate, when doing a Gram stain a second and a third time, ended up seeing something entirely different: 

Alas, it was none other than contamination itself. Now, what was nice was that when he did his gram stain on one of the other tubes, we did have e coli, albeit some regions that were purple: 

But because of this, a plasmic extraction was done either way, as he also thought that he just didn't do a Gram stain correctly on the other slide because it was otherwise the shape of e coli and there were regions that were pink. 

What was interesting was that we first needed to find the source of our contamination, because when a Gram stain was done on the plate that had been taken from freeze back, the gram stain came out perfectly fine. However, when looking at our 2X LB (which had double the number of ingredients in it), and especially after having done a Gram stain on it, we verified that this was indeed the source of contamination. This likely happened because this LB that we made had double the number of ingredients that make it a very suitable environment for growth, and if anything had jumped in there when using it, or if our techniques hadn't been sterile enough, then it is possible that whatever made its home in the lb was able to flourish. Because of this as well, my own hypothesis is that whatever was in there overtook the E coli, which could explain why it did look like E coli but had readings of purple, and by the time my lab mate did that third Gram stain, it had had completely taken over. What will be doing next time to account for this is to make sure to make the 2x I'll be right before we are going to inoculate into this to lessen the amount of time that it stays out and to make sure that it is as fresh and as clean as possible.

On another note, a gel run was performed to check the size of the plasma that had been extracted from that E coli that had a bit of purple on it, and the plasmid size actually fit what we were looking for. The pRad1 plasmid is approximately 6,800 base pairs long, and when comparing to the 1kbp gel ladder, it was at the correct location to indicate that the plasma that had been extracted (and digested) was indeed the correct one.

On a much less likely note, it could be possible that the DNA we extracted is from another organism altogether that happens to be the same size as the one we are looking for, but again, this is probably not likely. For this, we can celebrate and move on to transformation starting Tuesday!

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 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

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