Paris/July 23
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Revision as of 23:04, 23 July 2007 by David.bikard (Talk | contribs)
Contents |
w121 growth
w121 culture launched on 22/07/07 did not grow ON: I had forgot to add DAP to LB-Erythro growth medium.
at 12h30, the following culture was launched:
- w121 in 2ml LB-Erythro-DAP
We were unable to perform growth kinetics analysis of w121 in different DAP-supplemented media (because the machine is occupied for the night
Transduction of DapA deletion in MG1655
- The transduction seems to have worked: isolation of clones on LB and LB+DAP for verification.
Migration of digestion products
MiniPrep
Digestion reactions
Each of the digestion reactions that follow were performed as follows:
- 20µL DNA solution (miniprep or PCR product)
- 5µL Buffer 10x
- 0.5µL BSA 100x
- 2µL of each of the 2 enzymes indicated
- H20 qsp 50µL
NEB3 buffer used for XbaI/PstI double digestion NEBEcoRI buffer used for both EcoRI/SpeI & EcoRI/PstI double digestion reactions
| Digestion Products | ||||||||
|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | size | Description | ||
| D22 | pSB1A2 open vector | BBa_J61047 (MP9.1 & MP9.2) | EcoI | PstI | Open pSB1A2 vector for BioBrick EcoRI/PstI insertio | |||
| D23 | AraC/pBad promoter fw-insert | BBa_I0500 (MP4.1 & MP4.2) | EcoI | SpeI | AraC/pBad promoter ready for use as a forward insert | |||
| D24 | lox71-ftsZ bw-insert | lox71-ftsZ PCR product P7 | XbaI | PstI | lox71-ftsZ(unmutated) ready for use as a backward insert | |||
| D25 | eCFP bw-insert | BBa_E0422 (MP6.1 & MP6.2) | XbaI | PstI | eCFP (RBS-OFR_LVA-T) ready for use as a backward insert | |||
| D26 | PoPs to GFP converter | BBa_E0421 (MP7.1 & MP7.2) | XbaI | PstI | PoPs to GFP converter ready for use as a backward insert | |||
| D27 | gfp-tripart | BBa_E0840 (MP8.1 & MP8.2) | XbaI | PstI | gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert | |||
PCRs
| PCR : Lox71-FtsA-FtsZ | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 2058 | |||
| 60 | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 3 Lox71-FtsA-F | 2.5µL | |||
| 3m00' | Oligo R 10µM | 2 FtsZ-R | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 35 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Toothpick in MG16655 Glycerol | |||||
| PCR : Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2 | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 2058 | |||
| 70°C (5x) without oligos + 60°C (35x) | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 3 Lox71-FtsA-F | 2.5µL | |||
| 3m00' | Oligo R 10µM | 2 FtsZ-R | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 40x | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | 8.5µl Lox71-FtsA-FtsZ-1 (~50ng) + 17.5µl FtsZ-2 (~50ng) | |||||
| PCR : B0030-DapAColi | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 2058 | |||
| 60°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | o20 RBS-DapAColi | 2.5µL | |||
| 3m00' | Oligo R 10µM | o7 DapAColi-R | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 35x | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Toothpick | |||||
| PCR : B0030-DapASubtilis | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 2058 | |||
| 60°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | o20 RBS-DapASubtilis | 2.5µL | |||
| 3m00' | Oligo R 10µM | o9 DapASubtilis-R | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 35x | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Toothpick | |||||
