User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/26

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September 26th, 2010

1. Run gel to verify PCR made on September 25th.

ColonyPCR 26Sep2010.JPG

Lanes: 1) Green ladder; 2) pSB3K3-J23101 + Δ RBS-GFP E0040 (colony 1, tube 1); 3) pSB3K3-J23101 + Δ RBS-GFP E0040 (colony 2, tube 2); 4) pSB3K3-J23101 + Δ RBS-GFP E0040 (colony 3, tube 3); 5) pSB3K3-Min. Blue Promoter + Δ RBS-GFP E0040 (colony 1, tube 4).


2. Make the following restrictions:

  • SpeI-PstI double restriction to BBa_J61002 plasmid containing promoter J23101.
  • SpeI restriction to pSB3K3 + J23101.
  • SpeI-PstI double restriction methods:
DNA -> 5 ul
Buffer 2 -> 2 ul (10% of total volume)
BSA -> 1 ul
SpeI -> 1 ul
PstI -> 1 ul
HPLC -> 10 ul (to complete total volume of 20ul)
Incubate at 37º C for 4 hrs.
  • SpeI Restriction methods:
DNA -> 5ul
Buffer 4 -> 2ul (10% of total volume)
BSA -> 1ul
SpeI -> 1ul
HPLC -> 11ul (to complete total volume of 20ul)
Incubate at 37ºC 4 hrs.