IGEM:MIT/2005/Experiments: Difference between revisions
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[[Image:Check.gif]] gel purify each | [[Image:Check.gif]] gel purify each | ||
[[Image:Check.gif]] ligate together | [[Image:Check.gif]] ligate together | ||
[[Image: | [[Image:Check.gif]] transform | ||
[[Image:Pending.gif]] identify correct clones (colony PCR, miniprep/redigest) <font color=red>ask TK about bb PCR primers</font> | [[Image:Pending.gif]] identify correct clones (colony PCR, miniprep/redigest) <font color=red>ask TK about bb PCR primers</font> | ||
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[[Image:Check.gif]] gel purify each | [[Image:Check.gif]] gel purify each | ||
[[Image:Check.gif]] ligate together | [[Image:Check.gif]] ligate together | ||
[[Image: | [[Image:Check.gif]] transform | ||
[[Image:Pending.gif]] identify correct clones | [[Image:Pending.gif]] identify correct clones | ||
Revision as of 07:12, 26 July 2005
[[../Experimental Flowchart/]]
Project Experiments
Input Device: [[../Input: Ligand#Experiments/|fluorescein]] Head Receiver Unit: [[../Receiver Head Unit: scFv#Experiments/|anti-fluorescein scFv]] Receiver 1: [[../Receiver 1: ToxR#Experiments/|ToxR]] Receiver 2: [[../Sensor 2#Experiments/|FecA]] Signal Processor: [[../Signal processing#Experiments/|Exp. Here]] Actuator: [[../Actuator#Experiments/|Exp. Here]]
indicates done! indicate pending...
Experiment Status
Needs Attention --get protocols from Jenny!
- FecA promoter PCR with higher annealing temperature to get a PCR product.
- PhoA PCR with higher annealing temperature to get less PCR products.
- MalE digest. Did not show on gel for purification.
scFvs: 4M5.3, S101A, 4420 biobrick ends digest scFv + pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones (colony PCR, miniprep/redigest) ask TK about bb PCR primers
MalE PCR digest MalE + pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones
PhoA PCR with higher annealing temperature digest PhoA+ pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones
FecA promoter PCR with higher annealing temperature digest FecApro + pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones
FecA (full length) PCR bb ends digest FecA + pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones
FecI PCR bb end digest FecI + pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones
FecR PCR bb end digest FecR + pSB1AK3-1 plasmid w/ E/S gel purify each ligate together transform identify correct clones
All overnights of bbs, miniprep plasmids Grow up overnights (W) and make glycerol freezer stocks (R) -- Jen -- see inventory page for updates
Experimental Timeline
- [[../July wk 3:/]]
1. Receive primers [math]\displaystyle{ \rightarrow }[/math] Make biobricks, with these [[../Techniques/]]
J07005 (phoA), J07006 (malE), J07012-14 (scFv 1-3), J07018 (FecA), J07019 (FecA promoter), FecR, FecI.
1.5 Make competent MC4100 cells, pour LB Kan plates, make freezer stocks of strains acquired from Registry
2. ToxR:
Can begin assembly of R0040::B0030, for use in parts J07024-28 (see August wk. 1)
3. Input Experiments : Verify that flourescin oligo's enter cells.
4. Assemble fecA promoter with GFP --> test (after J07019 complete)
- July wk 4:
FecA: Fuse scFv with FecA? ToxR:
- Aug wk 1:
ToxR: -Receive synthesized J07007 (ctx promoter), J07009 (ToxR') -Begin Assembly of: -J07011 (ctx promoter :: gfp reporter) -J07024-26 (promoter::RBS::ToxR'::scFv 1-3) -J07027-28 (Positive and Negative controls, promoter::RBS::ToxR'::PhoA/MalE)
- Aug wk 2:
- Aug wk 3:
- Aug wk 4:
- Sept wk 1