S-STEM Scholar Madison Jackson's Blog

Intro This week was largely dedicated to inoculations, with us also setting aside a flask to be measured for a growth curve. Media was made and plates were poured to ensure that we had enough for the rest of the semester. A lot of time was spent helping out Evan's group with inoculations, pouring plates, and gram staining for their various projects. Methods The process for gram staining is as follows: 1. clean station and gather materials 2.Clean slide with ethanol by applying ethanol to a kimwipe, then wiping the slide 3. Draw a circle with a wax pencil directly onto the slide, number the slide if multiple 4. Heat inoculation loop in flame until glowing then allow to cool To collect sample from beaker, wave beaker over flame to prevent contamination the use loop to collect and recap sample 5. Smear collected sample in the circle, then heat fix by passing the slide over the flame briefly. 6. Bring slide over to a sink, then flood plane with crystal violent and let sit for 1 minute ...

Intro Between me being sick and Alex being sick later in the week, there was  limited amount of progress that was made this week. With our time, we did manage to get another set of RNA extraction done with some minor modifications to our normal procedure in order to hopefully increase our overall yield at the end so that we can move forward with sequencing.   Methods Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes. RNA isolation procedure: 1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube 2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times without repetition (minor change) 3. centrifuge the tube for one minute t...

Intro This week we started with RNA extraction, then had to do another round of desiccation to, hopefully, get better numbers. We also discussed this week the make of our rehydration buffer, as the recipe has changed slightly and after that change we have seen a decline in our results. Methods Pre-desiccation cell packing  1. Normalize 3ml of culture to an OD between 0.95-1.00 2. Spin down 1ml of media, remove supernatant and resuspend pellet in nuclease free water 3. Resuspend pellet, then centrifuge and remove supernatant 4. Add the second ml of culture, repeat washing steps 5. Add the third ml of culture, repeat washing steps 6. Plate 100ul dots into 1 inch kapton squares in a 6 well plate in triplicate Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes. RN...