S-STEM Scholar Melanie Castaneda's Blog

  Introduction  Last week, we were able to clean up our left fragment and get it into the -20℃ freezer. It was the last fragment that we needed to clean in order to start assembling them together. The goal for this week is to do Gibson Assembly.  Methods  3/5/2024  I started off the week by making more media so that we could use it later on in the week or after spring break. Since our media would be going bad during spring break, I wanted to make new ones to use after assembly. I wouldn’t want older media to cause us to have unwanted results. I made 100 ml of TGY broth, 100 ml of LB broth, and 200 ml of LB + agar.  We also planned out how we will be plating the bacteria after assembly. We decided on having 8 plates total that will include a negative control, positive control, and two sets of experimentals. We will be doing four plates with no antibiotic and four plates with ampicillin.  3/6/2024 We poured plates to have them ready for after assem...

  Introduction  In previous weeks, we have not been getting successful results using the DNA Cleanup Kit for our left fragment. We have used the same kit to clean up the other three fragments. We decided to try an alternative way of cleaning up our left fragment by doing alcohol precipitation.    Methods 2/26/2024  We did alcohol precipitation to try to clean up the left fragment. We followed the following protocol: Add the following to your sample:  0.1 vols of 3 M sodium acetate  2.5-3.0 vols of ice cold 100% ethanol Vortex to mix thoroughly Precipitate at -20℃ overnight or -80℃ for 1 hour Centrifuge at full speed for 30 minutes  Wash pellet twice with 0.5 mL of ice cold 75% ethanol, spinning at 4℃ for 10 minutes each time Take ethanol out, spin quickly (10s top speed) to remove the trace amount of ethanol Air dry the pellet and resuspend in an appropriate volume of nuclease free water *precipitating small amounts of RNA* Glycogen 20 ng...