Team:Grenoble/Biology/Notebook/September

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iGEM Grenoble 2012

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September


Week 36: September 03rd to September 09th

Goal of the week:

We tested the double mutant strains construction (protocol).
We did some verification colony PCRs with HF Phusion enzyme (protocol) in order to check the constructions.

To separate (protocol) the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_28

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1:DNA ladder (Smart Ladder)
  • Lane 2: araC PCR product in WT strain
  • Lane 3: araC PCR product in ΔaraC ΔcyaA strains
  • Lane 4: envZ PCR product in WT strain
  • Lane 5: envZ PCR product in ΔenvZ ΔcyaA strains
  • Lane 6: cyaA PCR product in WT strain

photo_gel_29

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1:DNA ladder (Smart Ladder)
  • Lane 2: cyaA PCR product in ΔenvZ ΔcyaA strains
  • Lane 3: cyaA PCR product in ΔaraC ΔcyaA strains
  • Lane 4: envZ PCR product in ΔenvZ ΔcyaA strains
  • Lane 5: araC PCR product in ΔaraC ΔcyaA strains

As the primers used for these PCRs were designed into the gene sequences, when there is no DNA bands, it shows that the knockout was efficient.
The double mutant constructions were confirmed.

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