S-STEM Scholar Alex Hugenberg-Cox Blog

 We started the week off strong with a pre-desiccation procedure, then assisting Evan with a procedure, and finally going over our sequencing protocol.  We did pre-desiccation on our A B and C flasks grown last week, following the usual cell packing procedure and then moved them to the egg the following day. Friday they were removed from the vacuum as it was unable to hold pressure that long but we were unable to perform RNA iso, so they dehydrated over the weekend.  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 We were also able to help Evan with a twitch motility procedure, preparing the specialized plates with the unique molds and later helping

 We started the week with RNA isolation, then did a clean up kit followed by a gel and fluorometer analysis. We did the normal RNA isolation procedure, then after analyzing the results did a clean up the next day. We did a Midori green RNA gel ( 1%, 50ul gel with 5ul of Midori Green)  from those results, and analyzed the sample quality the next day. RNA: 1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube 2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 5 cycles of 1 minute on and 2 minutes on ice 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. Retain the flow through 5. Add an equal volume of ethanol (95-100%) and mix thoroughly 6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Discard the

 We started the week inoculating 3 plates and 3 flasks from freeze back, then later in the week did a pre-desiccation procedure and discussed more about what the RNA sequencing would look like.  For pre desiccation we did the usual procedure, let it dehydrate for two days then placed it in the Egg, which is our new desiccator. Unlike the old glass one, this one is sealed and vacuumed making it true desiccation. Dehydration is a process of removing water or moisture from a substance, typically through heat, air circulation, or other drying methods. In dehydration, some water remains within the material, and the substance typically retains some degree of moisture. True desiccation, on the other hand, is a more extreme form of water removal that aims to completely eliminate all moisture from a substance.  A vacuum is not absolutely necessary for true desiccation, but it can be an extremely effective method for removing moisture. In the desiccation process, a vacuum proves valuable by comp