User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/05/21

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May 21st, 2010

1. Re-run gel to verify DNA extraction from E. coli K12 made on may 19th by Augusto & Paz, gel was run 50 min at 90V.

Paz-Augusto DNA Extraction.JPG
Lanes: 1,4) Ladder; 2) Augusto’s DNA extraction; 3) Paz’s DNA extraction.
  • This gel confirms that DNA extraction failed.

2. I incubated E. coli K12 on 5ml of LB medium for 12 hrs. at 300 rpm, to extract DNA using Miguel’s protocol. I also went to ask Miguel about his protocol but, fortunately, he gave me DNA from E. coli K12, so I don’t have to do DNA extraction again. With the DNA Miguel gave me, I ran a gel to verify the prescence of genomic DNA. In the same gel I loaded Paz’s DNA extraction but now with 5ul of loading buffer. The results are presented in the next figure:

Miguel-Paz DNAExtraction 21May2010.JPG
Lanes: 1) Ladder; 2) Miguel’s DNA; 3) Paz’s DNA extraction with 5ul of load buffer; 4) Ladder.
*It seems there’s not DNA at all, but I will run PCR to check if I can amplify Blue Promoter.

3. PCR for Blue Promoter

  • The reaction for blue promoter was repeated 2 times with each of the DNA’s (Paz & Augusto’s extraction and Miguel’s gift), OGR primers were used as control.
  • Reactions are as follows:
BP-Miguel x2 (B-M1, B-M2)
BP-Paz x2 (B-P1, B-P2)
BP-Augusto x2 (B-A1, B-A2)
BP-Ctrl - x1 (B-C-)
OGR-Miguel x1 (O-M)
OGR-Paz x1 (O-P)
OGR-Augusto x1 (O-A)
OGR-Ctrl - x1 (O-C-)
Total number of reactions: 11