Ethanol Precipitation

February 11, 2024 Introduction The bulk of this week was dedicated to the process of ethanol precipitation. Based on the 260/230 numbers from the nanodrop last week, it was clear that a contamination clean up was necessary, which in this case we used the ethanol precipitation procedure. Also done this week was the making of media for the sonorensis project, along with inoculation on each type of plate at the end of the week. Methods The procedure for ethanol precipitation is as follows: 1. Add .1 vols. 3M sodium acetate 2.5-3 vols ICE COLD 100% ethanol 2 uL glyco blue Vortex to mix thoroughly 2. Precipitate (ppt.) at -20℃ for 1 hour or overnight or at -80℃ for 1 hour (overnight will give more ppt. If RNA amount is low) 3. Centrifuge at full speed (13000 rpm) and 4℃ for 30 minutes 4. Wash pellet twice with .5ml ICE COLD 75% ethanol, spinning at 4℃ for 10 minutes each time 5. Take ethanol out, spin quickly (10s top speed) to remove as much trace amounts of ethanol as you can 6. Air dry the pellet (10-15 min.) and resuspend in an appropriate volume of nuclease free water For small amounts of RNA: 20 ng glycogen per sample may be added to the RNA before precipitation to aid visualization when ppt. small amounts of RNA Add 1 uL of a 20mg/mL solution of glycogen (RNase, DNase free), then follow procedure as normal The procedure for inoculation is as follows: 1. Clean work surface and gather inoculation supplies (bunsen burner, lighter/striker, inoculation loop, plate to inoculate onto, and plate to take sample from) 2. Light burner and place loop in inner cone to decontaminate 3. Let loop cool (about 30 seconds) before collecting sample from old plate, then streak onto new plate quickly to avoid contamination 4. Close plate and let sit to try for about 30 minutes before placing in correct incubator For inculation from broth onto plate: collect sample from broth as a "bubble" in the loop, then streak as normal Results Here are the results given from the nanodrop:
Before precipitation, 260/230 measurements were sitting at 1.14 at the lowest, with the lowest post precipitation value being zero. Here are all of the plates poured for the sonorensis project:
Two of each of the 13 plate varieites was then inoculated in the hood, one with E. coli and one with deinococcus sonorensis, and then left in the incubator over the weekend. Discussion The ethanol precipitation protocol was used in order to clean up the RNA islolation results of the previous week. The numbers indicate though that not only was a large portion of the concentration of our RNA samples lost, but they were also introduced to a large amount of contamination through this process. The 260/230 ratio shows that a large amount of unwanted biologcal contaminants entered the samples during the course of the procedure. Whether this was due to contaminants being introduced by myself, Alex, or possibly through the ethanol or nuclease free water, it is clear that this sample was not usable and the procedure of RNA isolation would have to be done again to hopefully reach the qPCR stage. Conclusion As stated above, the numbers post-precipitation were too low to work with, so for the next week the plan going forward is to again go through with RNA isolation of E. coli and then to move forward with qPCR.

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