Sunday 2^nd October

Visualization

Digestion of GFP 1.9 PCR product from the 9th September

By Maxence & Victor

As we had several issues to clones GFP 1.9 in pSB1C3 by Gibson and digestion-ligation, GFP 1.9 PCR product from the 9th September was digested by restriction NdeI enzymes in order to verify if the template we used was the good one.

For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes NdeI as following:

• 2 µL of GFP 1.9 PCR product from the 9th September
• 1 µL of buffer orange
• 1 µL of restriction enzyme NdeI
• 6 µL of water

The mix was 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

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:

• 28 µL of template (pPS16_019 treated by XbaI & PstI)
• 13 µL of vector (pPS16_018 treated by SpeI & PstI)
• 9 µL of water
• 50 µL of isopropanol
• 5 µL of CH3COONa 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:

• 1.5 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme
• 12.5 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 Maxence & Victor

Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together:

• 6 µL of template (pPS16_009 treated by PstI & XbaI)
• 3 µL of vector (BbaB0015 treated by PstI & XbaI)
• 1.5 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme
• 3.5 µL of water

A control was done without template. The mix were incubated for 30 minutes at rooming temperature.

Gel of digestion and cleaned-up products

By Maxence & Victor

Result of the migration

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 Maxence & Victor

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.

Plasmids extraction 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 plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

• pPS16_018 (FKBP - GFP 10) clone 6
• pPS16_019 (FRB - GFP 11) clone 4
• pPS16_009 (GFP 1.9) clone 1