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

From OpenWetWare

Jump to: navigation, search

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!
M3D1 PCR products (T/R top gel, W/F bottom gel
M3D1 PCR products (T/R top gel, W/F bottom gel

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. green team -template, contaminated
  4. green team +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. yellow team -template, contaminated
  12. yellow team +template, good
  13. blue team +template, good

It seems like there was probably some contamination of the PCR master mix we used with template DNA (pRS406) before the Wed/Fri section set up their reactions. From looking at the gel image, why do you think I say this? Also, can you tell from the gel if the contamination issue affected the Tue/Thurs section at all?

Personal tools