IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-24: Difference between revisions
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==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!=== | ===SUCCESS!=== | ||
[[Image: lig_1024.jpg]] | [[Image: lig_1024.jpg|none|thumb|350px| Colony PCR of J04500 + KaiB]] | ||
*Chose colony 6 (liu). | *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. | |||
Latest revision as of 21:53, 28 October 2006
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.
- Collect a sample at 0.6OD; then dilute sample to 0.4OD and 0.18OD.
