Paris/July 14
From 2007.igem.org
(Difference between revisions)
David.bikard (Talk | contribs) (→PCRs) |
Nicolas C. (Talk | contribs) (→PCRs) |
||
| Line 44: | Line 44: | ||
== PCRs == | == PCRs == | ||
| - | {{ | + | {{Paris_PCR_0| Title = Lox71-FtsA-FtsZ-1 |
|Name= Lox71-FtsA-FtsZ-1 | |Name= Lox71-FtsA-FtsZ-1 | ||
|Annealing= 55°C | |Annealing= 55°C | ||
| Line 53: | Line 53: | ||
|dNTP= 10µM 1µL | |dNTP= 10µM 1µL | ||
|n_oligoF= 3 Lox71-FtsA-F | |n_oligoF= 3 Lox71-FtsA-F | ||
| - | |v_oligoF= | + | |v_oligoF= 2.5µL |
|n_oligoR= 4 DEcoR1-FtsZ-R | |n_oligoR= 4 DEcoR1-FtsZ-R | ||
| - | |v_oligoR= | + | |v_oligoR= 2.5µL |
|water= 34µL | |water= 34µL | ||
|pol= Phusion 0.5µL | |pol= Phusion 0.5µL | ||
|DNA= toothpick in glycerol stock of MG1655 | |DNA= toothpick in glycerol stock of MG1655 | ||
| + | |Size= | ||
| + | |Success= | ||
| + | |Image= | ||
| + | |Band= | ||
|}} | |}} | ||
| - | {{ | + | {{Paris_PCR_0| Title = FtsZ-2 |
|Name= FtsZ-2 | |Name= FtsZ-2 | ||
|Annealing= 55°C | |Annealing= 55°C | ||
| Line 76: | Line 80: | ||
|pol= Phusion 0.5µL | |pol= Phusion 0.5µL | ||
|DNA= toothpick in glycerol stock of MG1655 | |DNA= toothpick in glycerol stock of MG1655 | ||
| + | |Size= | ||
| + | |Success= | ||
| + | |Image= | ||
| + | |Band= | ||
|}} | |}} | ||
| - | {{ | + | {{Paris_PCR_0| Title = Lox66-DapAColi |
|Name= Lox66-DapAColi | |Name= Lox66-DapAColi | ||
|Annealing= 55°C | |Annealing= 55°C | ||
| Line 93: | Line 101: | ||
|pol= Phusion 0.5µL | |pol= Phusion 0.5µL | ||
|DNA= toothpick in glycerol stock of MG1655 | |DNA= toothpick in glycerol stock of MG1655 | ||
| + | |Size= | ||
| + | |Success= | ||
| + | |Image= | ||
| + | |Band= | ||
|}} | |}} | ||
Revision as of 16:48, 18 July 2007
Contents |
What to do ?
- Remove the plate of the transduction experiment and the transformation ones from the 37°C incubator (Upper-left shelf): If it has worked and if someones comes on Sunday:
- isolation of the clones from the transduction on petri-dishes (LB+DAP+Erm+Citrate)
- LB + antibio culture of the transformants (to make a glycerol stock and to do MiniPreps)
- Make a gel (0.8%) and migrate the PCR products
- Purify them
- Do the assembly PCR
If enough time (we can also wait to have the plasmids to do everything at the same time):
- Digestion of the purifications products with appropriate enzymes
- Purification
What has been done
- Several colonies of transduced MG1655 are obtained (transduction from dapA- strain w121):
- Isolation of 10 different colonies on LB Citrate Erythro DAP plates
- Seeding LB-Amp cultures of the following strains
- DH5alpha transformed with the plasmid carrying the Biobrick pJ23100
- DH5alpha transformed with the plasmid carrying the Biobrick B0015
these two ON cultures will allow us to perform (tomorrow):
- Minipreps to isolate the plasmids carrying the biobricks
- Glycerol stocks of the transformed strains
DGAT expressing E.coli
- After transformation of DH5alpha E.coli with pKS::DGAT plasmid, a transformant clone was grown ON in LB Erythromycin medium
The culture (using a toothpick)was deposited on several solid media for growth:
either LB or M9 minimal medium. +/- IPTG (inducer of DGAT expression, dgat gene being carried by pKS::DGAT plasmid) +/- Nile Red (fat detection dye) +/- oleate at 2 different concentrations.
This experiment aims at finding out if E.coli cells that express DGAT from Acinetobacter can incorporate oleate (a fatty acid, catalyse the reaction between a fatty acid and diacyl glycerol and finally accumulate fat in the form of triglyceride.
PCRs
| PCR : Lox71-FtsA-FtsZ-1 | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | ||||
| 55°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 3 Lox71-FtsA-F | 2.5µL | |||
| 2m00' | Oligo R 10µM | 4 DEcoR1-FtsZ-R | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µL | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | toothpick in glycerol stock of MG1655 | |||||
| PCR : FtsZ-2 | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | ||||
| 55°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 5 DEcoR1-FtsZ-F | 10µM 2.5µL | |||
| 2m00' | Oligo R 10µM | 2 FtsZ-R | 10µM 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µL | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | toothpick in glycerol stock of MG1655 | |||||
| PCR : Lox66-DapAColi | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | ||||
| 55°C | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | 6 Lox66-DapAColi-F | 10µM 2.5µL | |||
| 2m00' | Oligo R 10µM | 7 DapAColi-R | 10µM 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µL | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | toothpick in glycerol stock of MG1655 | |||||
