Monday 4th July
Visualization
By Caroline and Lea
The size of the gBlock 2.2 is 808bp and 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.
gBlocks transformation 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 carried out on the morning. 3 other transformations were performed with plasmids pPS16_007, pPS16_008 and pPS16_009 extracted on 30/06/16. The transformation protocol was followed increasing plasmids quantities (5µL instead of 1µL).
Afterward cells were spread on Petri dishes containing LB + Ampicillin (50µg/mL) + X-Gal + IPTG.
Digestion of plasmids
By Mathilde and Alice
The following plasmids were digested again with EcoRI and HindIII:
- 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.
pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration
| Component
|
Volume (µL)
|
| Digested DNA
|
20
|
| Loading buffer
|
3.3
|
The DNA concentration was still insufficient meaning the extraction steps were not efficient.
Bringing DNA closer
By Naiane and Laetitia
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- 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 K1372001 characterization
Preparation of salycilate at 10 mM
By Charlène, Naiane and Léa
0.07g of salycilic acid was diluted in 50mL of water. The pH was adjusted to 7 with NaOH.
Culture of BL21
By Lea and Caroline
2 colonies from each Petri dish were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL).
Then each solution was split into 3x150µL and 350µL of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture.
Salicylate (main solution at 10mM) was added to obtain the following concentrations :
| Plasmid(s)
|
K1372001
|
K1372001 + pcl_TAA
|
K1372001 + pcl_TAG
|
K1372001 + pcl_Tq
|
| Clone
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
1
|
2
|
| Salicylate concentration
|
0
|
30µM
|
1mM
|
0
|
30µM
|
1mM
|
0
|
30µM
|
1mM
|
0
|
30µM
|
1mM
|
