I led the students to SOD1 yeast sequences to work on ApE.
1) Find out where is the mutation in DNA sequences.
2) Find an RE that can distinguish WT from mutant.
3) Use the RE of your choice, describe digestion results of WT and mutant genes.
4) Translate the genes into proteins and identify the nature of the mutation.
5) Figure which gene this is using blastp.
I then worked on math assignment3 and 2.
At the end of the class, I explained transformation. The key is to how force cells to keep the plasmid. We need to make cells dependent on the plasmid, like people depndent on cigarettes. By using auxotrophic marker, cells without the HIS3 of plasmid will not live in the absence of histidine.
I then explained pSH44, the reporter plasmid and URA3 fused with micro-satellite. How FOA is converted to a toxic stuff by normal URA3 that kills the cell. Only when URA3 is disfunctional due to failed MSH2, can cells surive on FOA plates. This is negative selection. I then added TRP5 of pSH44 into the mix, explained what kind of media should be use for FOA plates.
See:
http://www.youtube.com/watch?v=NxLhXF_jS8o
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