Monday 3rd October
Lab work
Visualization
Colony PCR of 32 clones containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence & Caroline
For that purpose, 32 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
|
30 sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
|
| 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)
|
| GFP 1.9
|
1135
|
GEL 6
GEL 7
Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1
By Maxence & Caroline
Extracted pPS16_009 from the 2nd October was digested by several restriction enzymes in order to verify if the template we used was the good one.
For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following:
- 5 µL of GFP 1.9 (pPS16_009) clone 1
- 2 µL of buffer orange
- 2 µL of restriction enzyme NdeI
- 11 µL of water
Furthermore, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes BsaBI as following:
- 5 µL of GFP 1.9 (pPS16_009) clone 1
- 2 µL of buffer orange
- 2 µL of restriction enzyme BsaBI
- 11 µL of water
The mix were incubated for 30 minutes at 37°C.
Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6
By Maxence & Caroline
Extracted pPS16_019 and pPS16_018 from the 2nd October were digested by restriction enzymes EcoRI in order to verify the concentration.
For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following:
- 5 µL of plasmid
- 2 µL of buffer FD
- 2 µL of restriction enzyme EcoRI
- 11 µL of water
The mix were incubated for 15 minutes at 37°C.
Gel of digested products
By Maxence & Caroline
After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
GEL
