Talk:20.109(S07): Start-up expression engineering

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(Bottom row, Wed/Fri Section Samples)
Line 29: Line 29:
# empty
# empty
# ladder (???)
# ladder (???)
-
# -template
+
# -template, contaminated
-
# +template
+
# +template, good
# red team -template, contaminated
# red team -template, contaminated
# red team +template, good
# red team +template, good
Line 37: Line 37:
# pink team -template, contaminated
# pink team -template, contaminated
# pink team +template, good
# pink team +template, good
 +
# -template, contaminated
 +
# +template, good
 +
# +template, good

Revision as of 12:22, 7 April 2007

On Day 1 of Module 3, you did a few things:

  1. Learned about the SAGA chromatin-remodeling complex and chose a non-essential sub-unit of the SAGA complex to delete
  2. Designed primers to amplify the URA3 sequence with flanking sequence from upstream and downstream of the sub-unit chosen for deletion (On Day 2: This DNA sequence will be able to get into the nucleus of the yeast cells we are transforming after we have made them "competent" for DNA uptake. Once in the nucleus, the flanking sequences with homology to upstream and downstream sequence of your SAGA sub-unit will allow your DNA product from PCR to integrate into the yeast genome, via homologous recombination, kicking out your SAGA sub-unit sequence and replacing it with URA3 sequence)
  3. Set up PCRs with those primers to amplify the URA3 sequence off of the pRS406 plasmid DNA. I ran your PCR products out on a 1% agarose gel. The key for the lane loadings are below, so check out your results!

Image:Gel mod3day1 PCR products.JPG

Gel lane loading order

Top row, Tue/Thur Section Samples

  1. empty
  2. ladder (100bp)
  3. purple team 1
  4. purple team 2
  5. blue team -template, good
  6. blue team +template, good
  7. red team +template, good
  8. red team -template, good
  9. pink team -template, contaminated (?)
  10. pink team +template, good
  11. green team -template, good
  12. green team +template, good
  13. yellow team +template, good
  14. empty

Bottom row, Wed/Fri Section Samples

  1. empty
  2. ladder (???)
  3. -template, contaminated
  4. +template, good
  5. red team -template, contaminated
  6. red team +template, good
  7. purple team -template, contaminated
  8. purple team +template, good
  9. pink team -template, contaminated
  10. pink team +template, good
  11. -template, contaminated
  12. +template, good
  13. +template, good
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