IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-8

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Analysis of yesterday's {KaiA/B + bkb}\X-P digest


All the lanes have a band around 3400 bp, which is the length of an uncut J04450 device (RFP insert + bkb). We think that our backbone extraction from 2006-8-2 was contaminated with uncut J04450, which would dominate the subsequent ligations. Thus, we will rerun the 2006-8-2 digest for a longer time, and run the gel longer as well before extracting.

One piece of evidence against the above hypothesis is the observation that our KaiA + bkb and KaiB + bkb ligations do not fluoresce red, as one would expect if they had been transformed with uncut J04450.

Lanes 3, 5, and 8 appear to contain the KaiA insert (which should be 903 bp). However, we're not sure why 1) There's still a 3400 bp band in those lanes, and 2) There's no backbone (2000 bp) in those lanes. It's possible that these cultures were inoculated from several colonies, but we were careful about choosing single colonies.

To-do

  • Talk about Western blotting
  • Redo part of the 2006-8-7 {KaiA/B+bkb}\X-P digest
    • Digest the DNA that went into lane 3 (10uL) like we did yesterday, and run it against undigested sample (10uL DNA). Likewise for lane 8 and lane 12 and lane 16. Let the digest go for 6 h; use more enzyme.
      • The cultures are labeled KaiA+bkb #2, #7, #11, and KaiB+bkb #3.
  • Redo the 2006-8-2 J04450\X-S digest.
    • We should have leftover miniprepped J04450 ready for use
    • Run the digest for longer (6+ hrs)
    • Try varying the amount of restriction enzyme
    • When we run the digest on a gel, run undigested J04450 as well
      • Acts as a negative control
      • Provides some reassurance that the J04450 device is correct as it is written in the BB registry
  • Religate and transform low copy plasmid backbone with RFP (if yesterday's transformants don't grow up)
  • Analyze sequencing results from 400b band sequence (Peng)
  • Miniprep more of the KaiA+bkb culture that went into lanes 3, 8, 12, and 16 from yesterday's digest.
    • The cultures are labeled KaiA+bkb #2, #7, #11, and KaiB+bkb #3.
  • Perry's experiments
  • Look into putting LacIq on the plasmid
  • Ligate GFP with low-copy plasmid

Transformation

Transformed J04500 plasmid into Top10F' competent cells. Peng will plate the transformants tonight and probably update this.
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