S-STEM Scholar Marisa Ostos Blog

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....

As spring break has come to an end, it is now time to start working with transformation once more. With there being just two months left of school, I wanted to make sure I was on the right track, so I spent some time planning and brainstorming with Dr. Tuohy about how to organize the rest of the semester. Ideally, one transformation will be run each week, but depending on growth / availability / potential contamination, a number of factors can play into an ideal timeline.  Along with this, I created a 1.5% agarose gel that was eventually placed into the fridge (this gel will be available to do a gel run after any future extractions and plasmid lysis I do in the future). I typically make 1% gels consisting of the following materials:  30 mL 1X TAE Buffer  0.3 g Agarose  [mixed in a 125 mL Erlenmeyer flask] However, this time I made a 1.5% agarose gel for electrophoresis that was made of the following materials instead (same ingredients, different amount of Agarose):...