Thuersday 22nd September
Lab work
Visualization
Glycerol stocks of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
"By Maxence, Mahnaz & Coline"
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
"By Maxence, Mahnaz & Coline"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
- pPS16_022 (GFP 1.9) clone X
Digestion of plasmids extracted from clones X, X, X and X containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
"By Maxence, Mahnaz & Coline"
To assess if DH5a cells were transformed with the good plasmid, plasmid digestion was run with XbaI as following:
- 1 µL of extracted plasmid
- 1 µL of buffer
- 1 µL of restriction enzyme XbaI
- 7 µL of water
A control was done with 1 µL of non-digested plasmid.
GEL
Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
By Maxence, Mahnaz & Coline
A colony PCR was done for 16 clones from the 20th September. For that purpose, 16 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
- 2.5 µL DreamTaq Buffer
- 0.5 µL of dNTPs (10mM)
- 1 µL of each primer mix (10µM)
- 0.13 μl of DreamTaq Pol
PCR was performed as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
3 min
|
| 30 cycles
|
95°C
|
30 sec
|
| 48.4°C
|
30 sec
|
| 72°C
|
1min 30sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
|
| Primers
|
iPS168 and iPS169
|
After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| FRB - GFP 11 - GFP 1.9
|
1500
|
GEL
