Revision as of 16:47, 20 September 2016

Tuesday 30^th august

"By Mahnaz"

Plasmid pZA11 were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit

By Mahnaz

Two preparations were made :

the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 + 0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix
the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water

Gibson products were incubated 1h at 52°c.

By Mahnaz

The transformation was performed following the usual protocol with 2µL of plasmid.

50, 150 and 350µL of the transformed DH5α with the 1-2 Gibson assembly product were plated on 3 plates of LB + chloramphenicol (30µg/mL)
350 µL of each Gibson assembly control and transformation control were plated on 2 plates of B + chloramphenicol (30µg/mL)

Petri cultures were incubated at 37°c overnight.

@@ Line 8: / Line 8: @@
 Plasmid pZA11 were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit
+====Gibson Assembly of segments 1 and 2====
+''By Mahnaz''
+Two preparations were made :
+* the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 +  0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix
+* the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water
+Gibson products were incubated 1h at 52°c.
+====Transformation in DH5α with the 1-2 Gibson assembly product====
+''By Mahnaz''
+The transformation was performed following the usual [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|protocol]] with 2µL of plasmid.
+* 50, 150 and 350µL of the transformed DH5α with the 1-2 Gibson assembly product were plated on 3 plates of LB + chloramphenicol (30µg/mL)
+* 350 µL of each Gibson assembly control and transformation control were plated on 2 plates of B + chloramphenicol (30µg/mL)
+Petri cultures were incubated at 37°c overnight.
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