Week 8: 5^th August - 11^th August

Plasmid 3:

05/08/13
We started the week by doing the golden gates over again, meaning the GG 3 and GG 2 (2.1 with FurBS 1, 2.2 with FurBS2 and 2.3 with FurBS3). In fact, our previous control positive had some unwanted white spots (01/08/13), thus suggesting some contaminations during the transformation step. curretnmy waiting

06/08/13
We performed 6 golden gates, 3 for the second construction, 2 for the third construction and one for the controle plasmid. We prepared a mix for the 6+1 tubes :

• 7x 1,27 = 8,89 µl of 1A3 plasmide
• 7x 1,74 = 12,18 µL of terminator
• 7x 1,5 = 10,5 µL of T4 Buffer
• 7 x 0,5 = 3,5 µL of BSA
• 7 x 0,5 = 3,5 µL of T4 ligase

We then added:
Tube 1. =

• 0,76 µl of Andersen's promoter
• 0,76 µL of RBS
• 0,76 µl OF Fur BS
• 2,93 µL of sfGFP

Tube 2. =

• 0,76 µl of Andersen's promoter
• 0,76 µL of RBS
• 0,76 µl OF Fur BS
• 2,93 µL of sfGFP

Tube 3. =

• 0,76 µl of Andersen's promoter
• 0,76 µL of RBS
• 0,76 µl OF Fur BS
• 2,93 µL of sfGFP
• 4,28 µL of water

Tube 4. =

• 1,25 µl of pLac O
• µL of RBS-sfGFP
• 3,4 µL of water

Tube 5. =

• 1,25 µl of pLac O
• 2,05 µl of EntA
• 3,37 µL of EntD
• 2,07 µl of EntF
• 0,5 µL of water

Tube 6. = Controle Plasmid

• 0,76 µl of Andersen's promoter
• 0,76 µL of RBS
• 2,93 µl of sfGFP
• 4,28 µL of water

On the afternoon, we launch another golden gate for EntB/C/E Tube 7. =

• 1,27 µl of plasmid 1A3
• 1,5 µL of pLacO
• 0,49 µl of EntB
• 0,65 µL of EntC
• 1,74 µl of terminator
• 1,5 µL of Buffer T4 ligase
• 0,5 µl of T4 ligase
• 0,5 µL of Bsa 1
• 5,88 µL of water

Additionnally, we did the optimization of our PCR over with the temperature gradient for the annealing step. current waiting

09/08 We make 38 tube of Top 10

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