Wednesday 28th September
Visualization
Glycerol stocks of 8 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)
By Maxence & Mahnaz
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 7 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 10 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 11 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 2 (Gibson 3 fragments)
- pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 3 fragments)
- pPS16_018 (FKBP - GFP 10) clone 5 (Gibson 3 fragments)
- pPS16_018 (FKBP - GFP 10) clone 6 (Gibson 3 fragments)
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
"By Maxence & Mahnaz"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 7 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 10 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 11 (Gibson 2 fragments)
- pPS16_018 (FKBP - GFP 10) clone 2 (Gibson 3 fragments)
- pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 3 fragments)
- pPS16_018 (FKBP - GFP 10) clone 5 (Gibson 3 fragments)
- pPS16_018 (FKBP - GFP 10) clone 6 (Gibson 3 fragments)
NanoDrop Measurements
By Maxence & Manhaz
| Sample
|
Concentration (ng/µL)
|
| FKBP - GFP 10 clone 4 (Gibson 2 fragments)
|
31.05
|
| FKBP - GFP 10 clone 7 (Gibson 2 fragments)
|
271.52
|
| FKBP - GFP 10 clone 10 (Gibson 2 fragments)
|
62.18
|
| FKBP - GFP 10 clone 11 (Gibson 2 fragments)
|
244.56
|
| FKBP - GFP 10 clone 2 (Gibson 3 fragments)
|
171.08
|
| FKBP - GFP 10 clone 4 (Gibson 3 fragments)
|
420.69
|
| FKBP - GFP 10 clone 5 (Gibson 3 fragments)
|
88.42
|
| FKBP - GFP 10 clone 6 (Gibson 3 fragments)
|
43.97
|
By Maxence & Mahnaz
In order to verify if extracted plasmids contain FKBP - GFP 10, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids extracted today (clones 4, 7, 10 & 11 for 2 fragments Gibson and clones 2, 4, 4 & 6 for 3 fragments Gibson) were used.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer (5X)
- 0,5 µL of Phusion polymerase
- 32.5 µL of nuclease free water
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
Perform PCR as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
30sec
|
| 30 cycles
|
95°C
|
30sec
|
| 64.4°C
|
30sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
|
| Primers
|
iPS168 and iPS169
|
By Maxence & Mahnaz
In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids extracted the 27th Sepembter (clones 3, 4, 7 and 8) were used.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer (5X)
- 0,5 µL of Phusion polymerase
- 32.5 µL of nuclease free water
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
Perform PCR as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
30sec
|
| 30 cycles
|
95°C
|
30sec
|
| 64.4°C
|
30sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
|
| Primers
|
iPS168 and iPS169
|
Gel of PCR products
By Maxence & Manhaz
4 µL of PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| GFP 1.9
|
1135
|
| FKBP - GFP 10
|
1030
|
