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.

GEL SYLVIE 5

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.