Saturday 1st October
Visualization
Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1
By Maxence & Victor
As we had several issues to clones GFP 1.9 in pSB1C3 by Gibson and digestion-ligation, pPS16_009 was digested by BsaBI 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 BsaBI as following:
- 7 µL of GFP 1.9 (pPS16_009) clone 1
- 2 µL of buffer orange
- 2 µL of restriction enzyme BsaBI
- 9 µL of water
The mix were incubated for 30 minutes 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.
Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation
By Maxence & Victor
For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following:
- 10 µL of FRB - GFP 11 (pPS16_019) clone 4
- 2 µL of buffer FD
- 1 µL of restriction enzyme XbaI
- 1 µL of restriction enzyme PstI
- 6 µL of water
And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes SpeI & PstI as following:
- 10 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6
- 2 µL of buffer FD
- 1 µL of restriction enzyme SpeI
- 1 µL of restriction enzyme PstI
- 6 µL of water
The mix were incubated for 20 minutes 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 (pPS16_019 treated by XbaI & PstI)
|
727
|
| Vector digested (pPS16_018 treated by SpeI & PstI)
|
2784
|
The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation
By Maxence & Victor
For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes PstI & XbaI as following:
- 5 µL of GFP 1.9 PCR product from the 9th September
- 2 µL of buffer FD
- 1 µL of restriction enzyme PstI
- 1 µL of restriction enzyme XbaI
- 11 µL of water
Furthermore, Bba0015 plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI as following:
- 10 µL of Bba0015 plasmid
- 2 µL of buffer FD
- 1 µL of restriction enzyme PstI
- 1 µL of restriction enzyme XbaI
- 6 µL of water
The mix were incubated for 30 minutes hour at 37°C. Then, 2 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 PstI & XbaI)
|
900
|
| Vector digested (BbaB0015 treated by PstI & XbaI)
|
2000
|
The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Gel of digestion and cleaned-up products
By Maxence & Victor
Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1
By Maxence & Victor
The following clones were put on liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight:
- pPS16_018 (FKBP - GFP 10) clone 6
- pPS16_019 (FRB - GFP 11) clone 4
- pPS16_009 (GFP 1.9) clone 1
