Paris/July 18
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| + | [[Paris/July 17|yesterday]] -- [[Paris/July 19|tomorrow]] <br> | ||
| + | == Transduction of w121 strain == | ||
| + | Titration of the stock of phage from 12.7.7. : ~10<sup>9</sup> phages/ml : OK | ||
| + | <br> | ||
== FtsZ<sup>TS</sup> strain screening == | == FtsZ<sup>TS</sup> strain screening == | ||
We want to test these strains : | We want to test these strains : | ||
| Line 35: | Line 39: | ||
* 50µL of DAP 0.2 mM OK | * 50µL of DAP 0.2 mM OK | ||
* 50µL of DAP 50mM OK | * 50µL of DAP 50mM OK | ||
| + | |||
| + | ==PCRs== | ||
| + | |||
| + | {{Paris_PCR_0| Title = Assembly PCR Lox71-Fts1-FTsZ1 + FtsZ2 | ||
| + | |Annealing= 50, 55, 60, 65°C | ||
| + | |Elongation= 3m00' | ||
| + | |Cycles= 35 | ||
| + | |Buffer= 5x 10µL | ||
| + | |MgCl2= 10µM 0µL | ||
| + | |dNTP= 10µM 1µL | ||
| + | |n_oligoF= 3 Lox71-FtsA-F | ||
| + | |v_oligoF= 2.5µL | ||
| + | |n_oligoR= 2 FtsZ-R | ||
| + | |v_oligoR= 2.5µL | ||
| + | |water= 30µL | ||
| + | |pol= Phusion 0.5µL | ||
| + | |DNA= Lox71-FtsA-FtsZ1 2µL + FtsZ2 2µL | ||
| + | |Size=2580 | ||
| + | |Success=NO | ||
| + | |Image= | ||
| + | |Band= | ||
| + | |}} | ||
| + | |||
| + | == E.coli pKS::DGAT == | ||
| + | |||
| + | * After overnight culture (see [[Paris/July_17#Exponential_culture_of_E.coli_pKS::DGAT_and_microscopy|July 17]]), we do not observe significant triglyceride synthesis. | ||
| + | * We decide to test in solid culture and wait some days. E.coli transformed by pKS::DGAT and the negative control (E.coli transformed by part B0015) were spread on LB medium +- oleate 2mM +- IPTG (0.4mM). | ||
| + | * We try in M9 Minimum Medium (see [[Paris/PROTOCOLS#Solid_M9_Minimum_Medium|Protocols]]) replacing lactose/galactose by 2mM oleate, too. | ||
Latest revision as of 17:48, 7 October 2007
Contents |
Transduction of w121 strain
Titration of the stock of phage from 12.7.7. : ~109 phages/ml : OK
FtsZTS strain screening
We want to test these strains :
- 121.4
- 121.5
- 121.6
- 129
- 1.129
- DCR 14.1
- DCR 14.2
- DCR 14.3
I made glycerol stock of these strains, stored in the freezer. Test :
- Spread these strains on two plates (preheated before spreading) :
- One at 30°C
- One at 42°C
The results tomorrow.
Minipreps
We have 2 clones for each plasmid :
- E0422
- E0241
- E0840
- B0030
- J61047
Elution within 50µL H2O
DNA is within the Miniprep box in the -20°C fridge.
Digestion products
Photos !!!
DAP solution contamination test
Results :
- 50µL of H20 (control) OK
- 50µL of aliquot DAP 50mM (in use) OK
- 50µL of DAP 1mM CONTAMINATED
- 50µL of DAP 0.2 mM OK
- 50µL of DAP 50mM OK
PCRs
| PCR : Assembly PCR Lox71-Fts1-FTsZ1 + FtsZ2 | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 2580 | |||
| 50, 55, 60, 65°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 3 Lox71-FtsA-F | 2.5µL | NO | ||
| 3m00' | Oligo R 10µM | 2 FtsZ-R | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 30µL | [[Image:|30px]] | |||
| 35 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Lox71-FtsA-FtsZ1 2µL + FtsZ2 2µL | |||||
E.coli pKS::DGAT
- After overnight culture (see July 17), we do not observe significant triglyceride synthesis.
- We decide to test in solid culture and wait some days. E.coli transformed by pKS::DGAT and the negative control (E.coli transformed by part B0015) were spread on LB medium +- oleate 2mM +- IPTG (0.4mM).
- We try in M9 Minimum Medium (see Protocols) replacing lactose/galactose by 2mM oleate, too.
