E. coli RNA isolation and AMC

Introduction 
     As mentioned at the end of last week's post, the main focus of this week was completeing E. coli RNA isolation clean enough to be able to continue onward with qPCR. Seeing as Alex needed to be present for the RNA isolation and is busy running multiple projects, this wasn't able to be done until the end of the week. However, a good portion of time was dedicated to Alex teaching Carime and myself about the activated methyl cycle and the premise of our project, which will be discussed in detail at the end of this post.
Methods
    The procedure for RNA isolation is as follows: 1. Resuspend fresh or frozen pellet in 800uL RNA lysis buffer and transfer misxture into ZR BAshingBead Lysis tube (0.1 and 0.5mm) 2. Secure tube in bead beater fitted with 2mL tube hold assembly and process 3. Centrifuge tube for 1 minute to pellet debris (1 min. on, 2 min. off) 4. Transfer up to 400uL of cleared supernatant into a Zymo-Spin IIICG Column in a Collection Tube and centrifuge. Save the flow-through 5. To the flow-through, add an equal volume ethanol (95-100%) (1:1) and mix well 6. Transfer the mixture into a Zymo-Spin IICR Column in a Collection Tube and centrifuge. Discard the flow-through 7. Add 400uL RNA prep buffer to the column and centrifuge. Discard the flow-through 8. Add 700uL RNA wash buffer to the column and centrifuge, Discard the flow-through 9. Add 400uL RNA wash buffer and centrifuge the column for 1 min. to ensure complete removal of the wash buffer. The carefully transfer the column into a nuclease-free tube 10. Add 50uL DNase/RNase-Free Water directly to the column matrix and centrifuge. 
Results 
    Before the procedure, the samples taken had incubated for an hour and a half before being measure as .16(A), 1.05(B), and .09(C). Because of these numbers, it was decided that sample B would be used for the rest of the process going forward. The results for the procedure are shown here:
As shown here, numbers are bad and mistakes were made during the procedure; refer to discussion for further details. 
Discussion 
    For the RNA isolation procedure, Alex allowed me to handle it under constant surveillance. Unfortunately despite our best efforts I managed to add RNA lysis buffer in place of RNA wash buffer at step 8 of the procedure, completely throwing off any progress for the day. The samples were ruined, and would have to be taken again another week. 
Conclusion 
    Due to the mix up, the plan for next week again is to complete RNA isolation and begin qPCR proceedings. On my part, to avoid future errors like this one, I plan to double or triple-check labels whenever attempting a new procedure. AMC About half of this week's time was dedicated to discussing and learning about AMC, the activated methyl cycle. ALex gave Carime and myself the beginners guide to the process and our current project.
(Original notes and additional cleaner page for additional info pictured above) The point of the activated methyl cycle is to act as the cell's main methyl donor. The activated methyl cycle (or AMC for short) is typically shown as beginning with S-adenosyl methionine (SAM), which is toxic, so genes will code for a protein (mt-trans in this case) which changes SAM to S-adenosyl-L-homocysteine (SAH). SAH is converted to S-ribosylhomocystinase through the protein lux S. Lux S also produces DPD in a side reaction which relates to AI-2 and quorum sensing, or how cells communicate with each other. Homocysteine is converted to methionine by Met-E and Met-H. Mthionine is then converted to SAM by the protein Met-K. The cycle follows the circle shown in the images, with each substrate (SAM, SAH, SRH, etc.) becoming less toxic as the cycle continues. With each step there are slight amounts of substrates and proteins both lost and gained as they are used up around the cell. Some cells are capable of bypassing certain steps (going from SAH directly to homocysteine through the protein SAH-H). The cycle cannot stop at any given point because some steps are toxic. The process can't stop at the nontoxic points though, because the cell can;t effectively stockpile on any of the given substrates without needing more of something later as there's no way to predict exatly how much of anything the cell will need in 24 hours, much less an extended period. There's also assumed to be a bottleneck somewhere in this process which keeps too much of any given substrate from piling up. This is where the idea of our project comes in. By irradiating cells via UV, the proposed idea is that we will be able to pinpoint this bottle neck based on Met-K production post radiation.

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