S-STEM Scholar Madison Jackson's Blog

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...

Intro This week was slightly shortened as the lab was closed on Friday, 10/25. What we did do this week was start off with prepping samples for desiccation and put them into the desiccation chamber to sit over the course of the weekend. Samples were pulled out on Friday by Chad as he had access to the lab and removed the samples so they weren't too desiccated to be able to perform RNA isolation on them. This does mean, however, that the samples were allowed to sit for longer than normal total time. 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 Plates were moved into desiccator after 48 hours of drying time...

Intro This week involved another round of RNA isolation, followed by purification and then an RNA gel and fluorometer check to ensure quality of the RNA before proceeding.  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 3. centrifuge the tube for one minute to pellet debris 4. transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through 5. Add an equal volume of ethanol (95-100%) and mix well 6. tra...

Intro This week consisted of inoculating 3 new flasks and 3 new plates from the freezebacks created recently. We started pre-desiccation procedures again and further discussed what sequencing would look like and what kind of results we should expect to see/interpret. 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 Plates were moved into desiccator after 48 hours of drying time Results & Discussion We discussed more RNA sequencing and what exactly that process would look like. It begins with RNA extraction and is followed by multiple quality checks to ensure a lack of degradation. Next, purification focus...

Intro The entirety of this week was dedicated to RNA extraction, an RNA clean-up procedure on all of the samples, and running multiple RNA gels to verify results of said RNA extraction. Methods Note: control samples (D, E, F) were normalized the previous week, pelleted, and supernatant was removed before placing samples into -80°C freezer over the weekend to be ready for RNA extraction of control samples as well as test samples on Monday 9/30/24 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 3. centrifuge the tube for one minute to pellet debris 4. transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through 5. Add an equal volume of ethanol (95-100%) and mix well 6. transfer the mix...