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!



 * 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.