9 thoughts on “C40 – Ciecierska

  1. How would the northern/ western blot compare to the experiments you did and would they be more accurate with their outcome?

    1. The northern/western blot measures RNA or protein levels respectively, so this would take away a little bit of the middle work of needing to reverse transcribe the RNA we lysed from our cells back to cDNA. It could be more accurate because since it would take away those extra steps, it allows for less error and product loss.

    1. Honestly, all the little steps that could impact our last test to see if our gene indeed had increased expression after DNA damage. Also, making sure the correct amount and correct solvents were used in our PCR’s because we needed 12 tubes of different combinations of solvents and materials.

    1. We ended up using our Gene Specific forward and reverse primers that we created at the very beginning of the semester. They were used to see if our gene had increased expression after DNA damage was induced. We also used Rad-51 and Rpp0 primers as our controls.

    1. Thank you! So we had induced DNA damage, and in order to extract the RNA from those cells, we needed to break those cells apart. This also stopped the DNA damage process. We placed our +HU (with hydroxyurea) and UT (untreated) samples into a centrifuge, where we spun the cells at 3500 rpm for 1 minute. We then separated our cells from our media. (Using a pipet to take out the top layer that was the media). Next, we added 300uL of Lysis Buffer to each sample, and then pipetted up and down to mix and resuspend the cells in the buffer. Our samples were then stored at -20C in order to get them ready to have the RNA extracted. Hope that helped!

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