Monday 4th July
Lab work
Visualization
By Caroline and Lea
The size of the gBlock is 808bp so the ratio was 11.69 (>7).
GBlock was gently centrifugated and resuspended with 75µL of TE. The solution was then vortexed and stored at 50°C for 20 min. 1µL of ligase buffer, 1µL of ligase and 1µL of digested plasmids pUC19 were mixed with 7µL of gBlock 2.2. The resulting plasmid will be called pPS16_004.
2 control solutions were made:
- 1µL of digested plasmids pUC19 and 9µL of water
- 1µL of plasmid, 1µL of ligase and 1µL of water.
Transformation of Gblocks in DH5α
By Caroline and Lea
6 transformations were performed with the ligation products containing plasmids: pPS16_001, pPS16_002, pPS16_003, pPS16_005, pPS16_006 and #pPS16_004 created on the morning. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted on 30/06/16. TODO ajouter le lien The transformation protocol were followed increasing plasmids quantities (5µL instead of 1µL).
After transformation, cells are spread on petri dishes containing LB + Ampicillin (50µg/mL) + Xgal + IPTG.
Digestion of plasmids
By Mathilde and Alice
The following plasmids were digested again with EcoR1 and HindIII using this protocol:
- pPS16_001 (from the 6 clones that were selected on 29/06/16)
- pPS16_003 (from the 6 clones that were selected on 29/06/16)
- pPS16_005 (from the 6 clones that were selected on 29/06/16)
- pPS16_006 (from the 6 clones that were selected on 29/06/16)
| Component
|
Volume (µL)
|
| Plasmid
|
10
|
| Red buffer 10x
|
2
|
| Water
|
7
|
| EcoRI enzyme
|
0.5
|
| HindIII enzyme
|
0.5
|
The digestion products were mixed as follow to be migrated on an agarose gel.
| Component
|
Volume (µL)
|
| Digested DNA
|
20
|
| Loading buffer
|
3.3
|
The DNA concentration was still insufficient meaning the extraction step was not efficient.
Bringing DNA closer
By Naiane and Laetitia
Plasmids from the following strains were extracted with the kit "Charge Switch-Pro Plasmid Miniprep":
- DS-NMcas
- DS-SPcasN-
- DS-ST1casN-
- DS-TDcasN-
Then plasmids were digested with AvrII
| Component
|
Volume (µL)
|
| Plasmid
|
10
|
| Tango buffer 10x
|
2
|
| Water
|
7
|
| AvrII enzyme
|
1
|
The mix was incubated at 37°C for 1 hour.
An electrophoresis was done (0.8% of agarose).
| Component
|
Volume (µL)
|
| Digested DNA
|
5
|
| Water
|
5
|
| Loading buffer
|
2
|
Biobrick K13 characterization
Culture of BL
2 colonies from each petri dishes cultured in 500µL of LB, streptomycin(500µg/mL) and chloramphenicol (30µg/mL).
Then each solution were split into 3 parts of 150µL.
Each aliquot is composed of different concentration of salycilate (main solution at 10mM) :
| Concentration of salycilate
|
Volume of main solution
|
| 00 M
|
00 µL
|
| 30 µM
|
1.5 µL
|
| 1 µM
|
50 µL
|
For each colony from each condition (KB72001, KB72001 + PCLTAA, KB72001 + PLCTAG, KB72001 + PLCTq),
350µL of mix of LB + streptomycin + chloramphenicol were added to a culture of 500µL of LB streptomycin (50µg/mL).
Preparation of salycilate at 10 mM
By Charlène, Naiane and Léa
0.07g of salycilate was diluted in 50mL of water. The pH was adjusted to 7 with NaOH.7
