Saturday 1^st October

Lab work

Visualization

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 :

GEL

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, Bba plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI as following:

• 10 µL of Bba plasmid
• 2 µL of buffer FD
• 1 µL of restriction enzyme EcoRI
• 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 :

GEL

The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.