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Crystal Violet and Twitch Motility Assays

Introduction: This blog is both for the full week Nov 18-22 as well as the three days before thanksgiving break. We were able to do a full pre-desiccation to RNA isolation procedure as well as help with a number of various procedures in the lab. This Blog is covering the other procedures done this week in lab.  We were  able to get our working and permanent freeze backs from the A, B, C, D and E plates inoculated last week. On the twenty-second, after the RNA isolation, five flasks were inoculated from each of the working freeze backs. After growing over the weekend, we noticed a difference from the normal growth.  Usually the flasks end with a small amount of growth in the media and a large ball/clump of cells gathered. This was thought to be due to the movement, however others notated that in all the previous semesters when they were working with  deserti , it grew homogenously with none of the clumps we have been getting that have continuously worsened over the semester. When we sta
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Pre-desiccation to RNA isolation Procedure

Introduction This blog is both for the full week Nov 18-22 as well as the three days before thanksgiving break. We were able to do a full pre-desiccation to RNA isolation procedure as well as help with a number of various procedures in the lab. This Blog is covering the pre-desiccation to RNA isolation procedure. Procedure We started with our pre-desiccation on monday. From our flasks A B C D and E made last week, all except C were clear of contamination and able to go through desiccation. C was a yellow color with clear physical differences compared to the other flasks, as they all had the usual clumping and normal off-white color. We continued to pre-desiccation with the four flasks.  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 washin
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Growth curves and Freeze backs

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This week was mostly full of inoculations. We started the week both with new growth plates as well as a flask for a growth curve, and were able to help out Evans group with some inoculations later in the day as well. We both made some media and poured some plates to make sure we have enough to finish the semester. We wanted to get a growth curve to make sure we were growing for the optimal amount of time. Bacterial populations grow in a pattern that we can see in a graph and this pattern includes special phases: lag, exponential, stationary and death. During the lag phase, bacteria prepare for reproduction and change to new ecological conditions by synthesizing enzymes. In the exponential phase, cells divide quickly and the population doubles consistently at set intervals. In the stationary phase, growth rates drop as nutrients run low and waste builds up. When the population starts to decline because of resource depletion, and the buildup of toxic metabolites, it is during this period
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RNA Isolation

  This week was a short week for me as I was sick, with RNA isolation and normal project supplies upkeep being the only things done as well as general assistance of other groups. Monday we performed RNA isolation. The samples were grown and prepped last week, and had undergone 2 days of dehydration and 3 days of desiccation.  After other groups success with less bead beating time, and very high yields of RNA we also lowered our bead beating time yet saw no difference except all of our results were of low yield as opposed to one out of three. We also heated the nuclease free water to dilute in to 60 degrees Celsius to help with the binding efficiency and make it so that less RNA is lost.  Heating elution can significantly improve RNA yield through several key mechanisms. Higher temperatures help break down complex RNA secondary structures, increasing the molecule's solubility and making it easier to extract from binding surfaces. By partially denaturing RNA, heat reduces hydrogen bo
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Isolation and Desiccation

 We started this week off with an RNA Isolation, then prepared for and performed a pre-desiccation procedure later on. We did the usual RNA isolation procedures on samples A B and C. Last week the samples were prepared then moved to the Egg for true desiccation after 24 hours of drying. They then underwent true desiccation for 6 days before rehydration and RNA isolation. The wells were rehydrated using 650ul of the rehydration buffer, then put on he shaker for 15 minutes. After that we used inoculation loops to scrape the cells before centrifuging them to pellet for the RNA isolation procedure. 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 c
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Reviewing Sequencing Procedure

 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
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RNA Clean up

 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
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