Tuesday 11th October
Visualization
Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation
By Sylvie
For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following:
- 6 µL of FRB - GFP 11 (pPS16_019) clone 4
- 4 µL of buffer FD
- 1 µL of restriction enzyme XbaI
- 1 µL of restriction enzyme PstI
- 28 µL of water
And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:
- 3 µL of FKBP - GFP 10 (pPS16_018) clone 6
- 3 µL of buffer FD
- 1 µL of restriction enzyme SpeI
- 1 µL of restriction enzyme PstI
- 1 µL of alkaline phosphatase FastAP
- 21 µL of water
Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation
By Sylvie
For that purpose, GFP 1.9 PCR product from the 4th October was cut by restriction enzymes PstI & XbaI as following:
- 5 µL of GFP 1.9 PCR product from the 4th October
- 3 µL of buffer FD
- 1.5 µL of restriction enzyme PstI
- 1.5 µL of restriction enzyme XbaI
- 20 µL of water
Furthermore, Bba0015 plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI as following:
- 3 µL of Bba0015 plasmid
- 3 µL of buffer FD
- 1 µL of restriction enzyme PstI
- 1 µL of restriction enzyme XbaI
- 22 µL of water
And a control was also digested in order to verify the activity of restriction enzyme SpeI:
- 2 µL of FKBP - GFP 10 (pPS16_018) clone 6
- 2 µL of buffer FD
- 1 µL of restriction enzyme SpeI
- 15 µL of water
The mix were incubated for 20 minutes at 37°C.
GEL of digestion and cleaned-up products
By Sylvie
After each digestion, 5 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
|
| Vector digested (BbaB0015 treated by PstI & XbaI)
|
2000
|
The digestions were let at 37°C for an additional 45 minutes.
The FRB - GFP 11 (pPS16_019) clone 4 digestion products were put on gel and then cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.
The others digestion products (FKBP - GFP 10 (pPS16_018), GFP 1.9 PCR product and Bba0015 plasmid) were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. Then the purification products were put on gel in order to assess their quantity.
Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation
By Sylvie
Once template and vector were cut, they were mix together and were precipitated by ethanol as they were not concentrated enough. The ethanol precipitation was run as following:
- 45 µL of template (pPS16_019 treated by XbaI & PstI)
- 5 µL of vector (pPS16_018 treated by SpeI & PstI)
- 100 µL of EtOH 100%
- 5 µL of CH3COONa 3.3M
The mix was put 30 minutes at -20°C and was then centrifugated 10 minutes at 11 000 rpm and 4°C. The supernatant was removed and the pellet was washed twice with 100 µL of EtOH 70%. Finaly, the pullet was dried.
DNA ligase was used to join the sticky ends of the template and vector together:
- 2 µL of Buffer T4 10X
- 0.5 µL of ligase T4 enzyme
- 17 µL of water
A control was done with digested plasmid alone:
- 5 µL of pPS16_018 treated by SpeI & PstI
- 2 µL of Buffer T4 10X
- 0.5 µL of ligase T4 enzyme
- 12 µL of water
The mix were incubated for 30 minutes at rooming temperature.
Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation
By Sylvie
Once template and vector were cut, they were mix together with water and were precipitated by ethanol as they were not concentrated enough. The ethanol precipitation was run as following:
- 20 µL of template (GFP 1.9 treated by PstI & XbaI)
- 12 µL of vector (BbaB0015 treated by PstI & XbaI)
- 18 µL of water
- 100 µL of EtOH 100%
- 5 µL of CH3COONa 3.3M
The mix was put 30 minutes at -20°C and was then centrifugated 10 minutes at 11 000 rpm and 4°C. The supernatant was removed and the pellet was washed twice with 100 µL of EtOH 70%. Finaly, the pullet was dried.
DNA ligase was used to join the sticky ends of the template and vector together:
- 2 µL of Buffer T4 10X
- 0.5 µL of ligase T4 enzyme
- 17 µL of water
A control was done with digested plasmid alone:
- 12 µL of BbaB0015 treated by PstI & XbaI
- 2 µL of Buffer T4 10X
- 0.5 µL of ligase T4 enzyme
- 5 µL of water
The mix were incubated for 30 minutes at rooming temperature.
Transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation
By Sylvie
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021), and with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual protocol.
BioBrick K2039000 and K2039001 characterization
Transfert of protein extracted from FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594
By Philippe
The proteins on the electrophoresis gel were transfered on blotting membrane using the protein transfert protocol.
