IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-24

Results of transformation

Plate with many cells on it; picked 8 to PCR (see 2 days ago for PCR protocol, except extension time decreased to 3 min). Expected value is ~2650bp.

Also restreaked; marked in chinese numerals :D in the fridge.

SUCCESS!

Colony PCR of J04500 + KaiB

• Chose colony 6 (liu).

Review

We have (based on old numbering system):

• J36000: KaiC on geneart plasmid
  • Sequenced
• J36001: KaiB on geneart plasmid
  • Sequenced
• J36002: KaiA on geneart plasmid
  • Sequenced
• J36010: Lac + RBS + KaiC (eg. J4500/sp + J36000/xp)
  • Sequenced
  • Frozen stock called "M2-7"
  • Kan / Amp
• J36011: Lac + RBS + KaiB (eg. J4500/sp + J36001 /xp)
  • Sequencing submitted, pending
  • Frozen stock called "colony E"
  • Kan / Amp
• J36012: Lac + RBS + KaiA (eg. J4500/sp + J36001 /xp)
  • Sequenced
  • Frozen stock called "A11" (I think, though I need to double check)
  • Kan / Amp
• J36021: J36011/sp + J36010 /xp
  • Sequencing not yet submitted
  • Frozen stock not yet made
  • Picked colony liu (6).
  • Kan / Amp
• J36022: J36012/sp + J36010 /xp
  • Sequencing submitted, pending
  • Frozen stock called "colony 1"
  • Kan / Amp
• J04430: GFP device behind Lac
  • Nick made, demonstrated works
  • ONLY Amp

Western blot outline

We are going to do the following "lanes:"

 1: J36010 in Top10, 0.4OD
 2: J36021 in Top10, 0.4OD
 3: J36022 in Top10, 0.4OD

 4: J36010 in Top10, 0.18OD
 5: J36021 in Top10, 0.18OD
 6: J36022 in Top10, 0.18OD

 7: J36010 in Top10, 0.6OD*
 8: J36021 in Top10, 0.6OD*
 9: J36022 in Top10, 0.6OD*

 10: GFP device in Top10, 0.4OD
 11: Negative control (H20? :D)
 12: Nothing

 *This OD value must be greater than 0.4 but less than stationary phase; any number works as long as all 3 lanes are the same

It is ambiguous as to what nm range the OD should be taken; I'd imagine 600nm through literature searches? The Endy protocol does not say the OD wavelength.

To secure the OD measurement, I assume one should:

• Either grow a colony to saturation or innoculate one from frozen
• Wait for the colony to reach ~0.6OD (DO NOT let it go above though, or it hits log phase)
  • Collect a sample at 0.6OD; then dilute sample to 0.4OD and 0.18OD.
    • 1uL sample, spin down, remove LB, and freeze.