Wednesday 12^th October

Visualization

Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 PCR product

By Sylvie

Extracted pPS16_019 and pPS16_018 from the 10 th October and the GFP 1.9 PCR product from the 4th October were digested by several restriction enzymes:

• 6 µL of FRB - GFP 11 in pSB1C3 (pPS16_019)
• 4 µL of buffer Tango
• 2 µL of restriction enzyme PstI
• 1 µL of restriction enzyme XbaI
• 1 µL of restriction enzyme NcoI
• 26 µL of water

And:

• 2 µL of GFP 1.9 PCR product
• 2 µL of buffer Orange
• 1 µL of restriction enzyme NdeI
• 15 µL of water

A control was done with SpeI and the mix were incubated for 2 hours at 37°C.

Gel of digested products

By Sylvie

After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Result of the migration

The results for GFP 1.9 could not be interpreted, the other results were good.

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

• 11 µL of template (pPS16_019 treated by XbaI, NcoI & PstI)
• 5 µL of vector (pPS16_018 treated by SpeI)
• 2 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme
• 1 µL of water

The mix was 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, DNA ligase was used to join the sticky ends of the template and vector together:

• 1 µL of template (GFP 1.9 treated by NdeI)
• 1 µL of vector (BbaB0015 treated by PstI & XbaI)
• 1 µL of Buffer T4 10X
• 1 µL of ligase T4 enzyme
• 6 µL of water

The mix was 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.

Liquid culture of FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 transformed the 11th October

By Sylvie

Few clones were obtained for the transformation done yesterday. Clones were selected and put on liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight.