S-STEM Scholar Melanie Castaneda's Blog

  Introduction:  In previous weeks, we have not been able to get good results when cleaning our D. rad PCR product using the Monarch RNA/DNA Cleanup Kit. We have been able to clean up our other three fragments using the kit but we were not able to get the desired results when cleaning up our left fragment. Jonathan suggested that we should look into an alternative method of cleaning up our sample: Alcohol Precipitation.  Methods:  2/22/2024  In order to be able to do alcohol precipitation, we had to run another PCR reaction using our D. rad genomic extraction sample to get our left fragment. We wanted to yield a higher volume of our sample compared to previous PCR reactions that we have run because we will lose volume when doing Alcohol Precipitation.  We mixed 36 μL of mastermix with 4 μL of our sample. We used two different genomic extraction samples labeled “MC” and MC2”. These samples had our highest ng/μL, A260/A280, and A260/A230 values. The Therm...

Introduction:  The main goal for this week was to finish acquiring all of the fragments and pieces that we need in order to move on to Gibson Assembly. We need clean PCR products of both of our left fragment and our pRHAM. Methods:  2/12/2024 We ran two different gels so that we could confirm that we had extracted the pRHAM plasmid and also successfully got the left fragment from our D.rad PCR. To do this, we made two separate 1% agarose gels and set them up in two separate apparatuses. For the pRHAM gel, we pipetted our 1,000 bp ladder into the first well. Then, we mixed 10 μL of our plasmid sample with 2 μL of UV dye. Since we had four total reactions when we did the plasmid extraction, we loaded four wells of our sample. This gave us a total of five wells.  For our D.rad PCR product, we loaded our 100 bp ladder into the first well. We had four total reactions from when we did the genomic extraction so loaded four more wells. Each well contained 10 μL of our D....

  Introduction:  After getting unwanted results during last week’s PCR cleanup, we needed to try another round of cleanup on our duplicate samples to see what our next steps would be. If the second PCR cleanup goes well, we can move forward with Gibson Assembly. If it shows similar results to last week’s, we will have to go back to the extraction process. Methods:  2/5/2024 We did our second PCR product cleanup on our duplicate samples for our Left Fragment and our pRHAM plasmid extraction. We followed the same procedure as last Thursday but we made a small change. Instead of adding 6 μL of DNA Elution Buffer, we added 10 μL.  2/7/2024 We were not able to get our materials to do the pRHAM plasmid extractions so we pushed it back a day. Instead, we were able to make freezebacs of our E.coli pRHAM culture. We made three freezebacs and put them in the -80°C freezer for long term storage.  As housekeeping, we inoculated D. radiodurans from one of our plates in...