S-STEM Scholar Jo Martin's Blog

  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...