S-STEM Scholar Jo Martin's Blog

 Week 10 November 25th - November 27th After running our big gel, we were left feeling a bit confused as to what the results were. It seems like no digested DNA moved at all, although the non-digested one did move. The ladder ended up showing completely, though. We ultimately decided to come back to this by deciding to choose a smaller DNA fragment that has less base pairs than Lambda DNA does. Since Lambda has such a high number of base pairs (48,502) and the xbar location is so split down the middle, it makes it difficult to see it accurately on our gel. Instead of trying for a third time, we decided to ultimately pick something smaller. We're still in the process of picking out a different type of DNA. The remainder of the week was spent assisting others with their projects. I worked alongside Vann and gram stained, inoculated, and ran a gel to confirm pRAD (6809 base pairs). We also spent time talking about her project and figured out the details.  Gel Results L1 : KB Ladd...

  Week 9 Nov 18- Nov 22nd Reflecting on last week, we’re at a point where we know that our Lambda DNA did not digest. We had quite a bit of independent time this week and most group members worked on their personal projects, in which I assisted with very briefly. For instance, we did an extraction on 11/19 of E. Coli and pRad that went extremely well. ng/ul     A260/A280   A260/A230 350.2     1.88    2.03 486.2      1.89   2.08 We also did gram stains and plasmid extraction on 11/21 that initially did not go well due to our user error on the very last step. After redoing it, the OD values were still poor. There’s discussion if the actual strain of E. Coli we have is not what we think it is since our plasmid extractions are turning out continuously poor and dirty. We also decided to redo our big gel on digested lambda, which we had done last time. Last week, when we ran our gel, although we ran it for almost two hours...

  Week 8 (Nov 11th- Nov15th) This week, we began the discussion of using lambda DNA to experience with as a control group. Because our past processes using xbar has been unsuccessful, we have decided to use experimental DNA in order to go through a step-by-step sleuth to see which step is failing. There’s also the possibilities that our ligation is not working but digestion is, our primers are bad, and so forth. Essentially, the major question is: is it us, or our processes? We decided that it was significant to keep our processes with lambda DNA as exact as possible in order to be able to compare the methods the most accurately. If the processes are not constant like before, there’s no point at all in doing the experiment. Lambda key data:   Lambda DNA: #N3011S, New England BioLabs 500 ug/ml 48,502 base pairs Xbar site at 24508 which leaves a 23994 bp difference The biggest issue with this is that the base pair difference is so tiny, sitting only at a 514 difference, th...

  Week 7 (Nov 4th-Nov 8th) This week, we concluded that our gram stain of pdCas9 and e. coli showed contamination which we were worried was staff. We decided to gram stain our parent plate and another member of the lab’s plate and decided if that second plate shows no contamination, we’ll inoculate onto two chloramphenicol plates. Plate A (parent plate) looked contaminated and Plate B (other lab plate) looked okay. The next day, the 5th, we decided to gram stain the two e coli plates we inoculated and found that whatever it was, it was not the e coli we were looking for. Generally, we have no idea what happened but it wasn't e coli with pdcas 9 and instead something completely else than we initially thought it was, although the bacteria is still unknown. We decided to take an old plate of pdcas 9 e coli and run a gel on that using 2 ul of dna, 8 ul of water, and 2 ul of dye at a concentration of 42.4 ng/ul at 50 amps.  On the sixth, we performed a transformation of pdcas9 and ...

Week 6 (Oct 28th-Nov 1st) On the 28th, we single digested pgK with xbar, inoculated our TGY and LB plates. We also gram stained one of our sonora plates, which looked okay. We decided to throw away our previous transformation tubes since there was no growth. We ended up ligating our pgRNa and Kana on the 30th using a 5:1 ratio. Typically, we used a 3:1 ratio, but increasing the insert will give it a better chance of ligating since we’ve been having issues in the past.  Quick T4 DNA Ligase: 1 ul 2X Quick Ligase Buffer: 10 ul  Vector DNA: 2 ul (pgRNA) Insert DNA: 3.5 ul (Kana) PCR Water: 3.5 ul ——— Total of 20 ul On the 31st, we ran a gel on our previously ligated pgRNA and Kana. If our ligation has not worked, we will then proceed with using lambda DNA as a control group to be able to understand what is going wrong: digestion, ligation, the primers, or even the DNA itself. For our ligation gel, we used 10 ul of pgK and 2ul of dye. The pgK on the gel showed us that the ligation ...

  Week Five (Oct 21-25) We started the week by gram-staining our pdCas9 and E. coli from 10/21. My first thought was that the actual gram stain result was poor, but I needed to use more ethanol for proper viewing, which was then redone. While I redid this to use more ethanol, we began working on a gel. The rest of the day was spent discussing microscope techniques along with different types of streaks for innoculation. For a four-way streak , you only pick up your cells ONCE. it’s important to do it quickly and avoid contamination as much as possible. It’s recommended to do it upside down to avoid contamination but, understandably, not everyone is capable of doing it that way, just do your best to avoid contamination and pushing air into the agar which could contaminate After our inoculation, we put the original plate into the fridge and the new plate in the incubator. We also originally had planned to use one R2A plate and one TGY plate but used two TGY plates as we were all out o...

  Week Four (Oct 14-18) We started the week by performing a plasmid extraction that we still need to confirm with a gel. Additionally, we also still did not reach a conclusion as to what happened revolving the previous gel we had done on our pgK. We think that the wrong ladder was used which caused everything to go wrong since we don’t know what ladder it is, which is why it’s so essential to write and label everything down. Afterwards, we went over lab math, how to pipette, how to load and run a gel, and doing a digest. SOP Loading a Ladder Start with largest volume In this case, we used 5 ul sample, 5 ul water, and 2 ul of dye We started with our 5 ul water for sample and 4ul into our ladder. Afterwards, we added our dye. When adding dye, submerge your pipette tip into the water so that the water is able to ‘grab’ onto the dye. Then, add the DNA. No DNA goes into the ladder since that’s what tells us the base pairs we are seeing. Pour TAE over the wells first. For this gel, we ra...

Week Three (Oct 7-11) On the day of the seventh, we discussed melting and plating LB broth with 500 ul of ampicillin. Since this was my first time doing any sort of work with media, we went over dating, how to plate, and the process of melting the media. For media: [Name of Media] [Amount of Media] [Date] [Initials] Ex: LB 250 ml 10/07 J.M. Keep LB broth at a heat of five and stir using heat glove to avoid boiling over This time, we plated half with plain LB broth and half with LB broth and 500 ul of ampicillin. On the eighth, we planned on putting out pgK on a gel to see bands. With this, we got successful ligation. After, we took a tube of pgRNA that we know had been cut by xbar successfully and added xbam to that. Additionally , on the ninth, we discussed how we previously took our digested kana and dosed it with xbar and was now planning on ligating and PCR. We also discussed the actual process of PCR. Step one: PCR is essentially targeted replication. The heat of our machine cause...

9/30-10/04 On the week of October 30th through November 4th, we made LB broth and prepared for transformation of e. Coli. We also did overnight ligation of our kanamycin and pgRNA since in the past, our ligations have failed. Used was the BioLabs quick ligation procedure. Quick ligation buffer (10ul) was used first as the buffer maintains the PH so the ligase doesn’t kill the pgRNA. 177.8 ng of kanamycin was needed according to the given calculator. 2 ul of 50 ng/u concentration of pgRNA; total of 100 ng needed. 3.5 ul of kanamycin of 50 ng/ul concentration needed for a total of 177.8 ng.

On September 24th, 2024, I began my first official week in the biotech lab. On my first day, the 25th, I spent the majority of my time learning about the specifications of deinococcus sonorensis, along with the key details of antibiotics we use, vectors, the digestion process, restriction enzymes, and so forth. This first week was generally about learning basic lab skills, such as how to make a 1% gel, understanding numbers for certain base pairs, and how to use a micropipette. As for lab work, on the 25th, we ran a gel on kanamycin at 50-60 milliamps for one hour. It was also noted that in order to have the most precise and correct results, it’s significant to ensure there are no air bubbles in the micropipette tip along with when you are inserting the actual tip, if you can very gently touch the wall of the gel, then you are in the well and are ready to deposit. The nanodrop read 74.1 ng/ul with an A260/A280 concentration of 1.84 and a A260/A230 concentration of 1.13.