Saturday 1^st 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