Tuesday 20th September
Visualization
Samples preparation for sequencing
"By Maxence, Mahnaz & Coline"
20 µL of plasmids pSB1C3 FKBP - GFP 10 (clones 7, 8, 9 and 10) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.
NanoDrop Measurements
"By Maxence, Mahnaz & Coline"
| Sample
|
Concentration (ng/µL)
|
| FKBP - GFP 10 clone 7
|
443.15
|
| FKBP - GFP 10 clone 8
|
260.26
|
| FKBP - GFP 10 clone 9
|
151.91
|
| FKBP - GFP 10 clone 10
|
230.11
|
| PCR product 1 obtained by iPS174 & iPS175
|
108.17
|
| PCR product 2 obtained by iPS173 & iPS176
|
136.91
|
| GFP 1.9 PCR product from 9th September (with DMSO)
|
420
|
| GFP 1.9 PCR product from 8th September
|
133
|
| FRB - GFP 11 - pSBI1C3 (pPS16_019) clone 4
|
98
|
Colony PCR of the 4 clones containing GFP 1.9 in pSB1C3 (pPS16_020) from the 12th September
By Maxence, Mahnaz & Coline
As results from sequencing were not good, clones sent previously and stocked in glycerol (2, 7, 8 and 12) were put on plate the 19th September, as each colony may not not be homogenous enough. Another colony PCR was done and anothers primers were used.
For that purpose, the 4 clones were 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
|
30sec
|
| 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 in pSB1C3
|
1135
|
No PCR products were obtained, plasmids were probably empty.
Gibson of cleaned up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI
By Maxence, Mahnaz & Coline
Gibson was performed with cleaned up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI, more precisely PCR product 1 obtained by iPS174 & iPS175 (plasmid) and PCR product 2 obtained by iPS173 & iPS176 (insert) with the following protocol:
For each 20μl of reaction, mix the following reagents :
- 0.63 µL of insert
- 1.2 µL of plasmid
- 8.17 µL of water
- 10 µL of buffer mix
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.
Transformation of DH5a cells with correct dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) obtained by Gibson
By Maxence, Mahnaz & Coline
Dh5a cells were transformed with corrected pSB1C3 containing dCas9 ST - GFP 11 (pPS16_017), or controls (no buffer mix and plasmid alone) using the usual protocol.
Cloning of GFP 1.9 from pUC19 (pPS16_009) in FRB - GFP 11 - pSB1C3 (pPS16_019) by digestion-ligation
By Maxence, Mahnaz & Coline
As difficulties were faced in order to obtain GFP 1.9 in pSB1C3 with Gibson strategy, we decided to construct first pPS16_022 (FRB - GFP 11 - GFP 1.9 in pSB1C3) and then clone FKBP - GFP 10 in pSB1C3 (pPS16_018) in it to construct pPS16_023 (FRB - GFP 11 - FKBP - GFP 10 - GFP 1.9 in pSB1C3).
For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes EcoRI & SpeI as following:
- 4 µL of GFP 1.9 PCR product from the 9th September
- 2 µL of buffer FD
- 2 µL of restriction enzyme EcoRI
- 2 µL of restriction enzyme SpeI
- 10 µL of water
And GFP 1.9 PCR product from the 8th September was cut by restriction enzymes EcoRI & SpeI as following:
- 10 µL of GFP 1.9 PCR product from the 8th September
- 2 µL of buffer FD
- 2 µL of restriction enzyme EcoRI
- 2 µL of restriction enzyme SpeI
- 4 µL of water
Furthermore, pPS16_019 was cut by restriction enzymes EcoRI & XbaI as following:
- 14 µL of pPS16_19 clone 4
- 2 µL of buffer FD
- 2 µL of restriction enzyme EcoRI
- 2 µL of restriction enzyme XbaI
The mix were incubated for 1 hour at 37°C. Then, 20 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Migration products expected were :
| Migration products
|
Expected band size (bp)
|
| Template digested (GFP 1.9 PCR product treated by EcoRI & SpeI)
|
900
|
| Vector digested (pPS16_019 treated by EcoRI & XbaI)
|
2900
|
The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol. Their concentrations were assessed by NanoDrop:
| Sample
|
Concentration (ng/µL)
|
| GFP 1.9 digested from the 8th
|
23
|
| GFP 1.9 digested from the 9th (DMSO)
|
103.5
|
| FRB - GFP 11 digested
|
18.67
|
Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together. Two protocols were used as template concentration was not the same between the two GFP 1.9:
- 5 µL of template (pPS16_009 treated by EcoRI & SpeI) from the 8th
- 7 µL of vector (pPS16_019 treated by EcoRI & XbaI)
- 2 µL of Buffer T4 10X
- 1 µL of ligase T4 enzyme
And:
- 1 µL of template (pPS16_009 treated by EcoRI & SpeI) from the 9th (DMSO)
- 7 µL of vector (pPS16_019 treated by EcoRI & XbaI)
- 2 µL of Buffer T4 10X
- 1 µL of ligase T4 enzyme
The mix were incubated for 1 hour at rooming temperature.
Transformation of DH5a cells with FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022) obtained by Digestion-Ligation
By Maxence, Mahnaz & Coline
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 - GFP 1.9 (pPS16_022), or controls (digested plasmid and water) using the usual protocol.
