User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/30

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June 30th, 2010

1. Replate transformations from June 29th, only transformations 2) p30-MinBP SpeI-PstI restriction + GFP BBa_K145015 XbaI-PstI restriction worked

  • Transformation 1) p30-MinBP SpeI-PstI restrictionn + GFP E0240 XbaI-PstI restriction a lot of colonies grew but we can´t distinguish which of them have the ligation of interest and which have the re-ligation of pSB1A2 (this plasmid contains the GFP that we want to use as reporter). I will extract this GFP E0240 by PCR and then make a restriction of this PCR product to ligate with p30-MinBP.

2. PCR to extract GFP E0240.

  • Primers used are Preffix FWD and Suffix REV.
  • Enzyme used: rTth.
  • A positive control I used a 1 kb biopart.
  • I will also test primers (the tubes marked in red are the old primers and the primers prepared on June 18th are the new ones).
  • Tubes were marked 1-4:
  1. GFP E0240 old primers
  2. GFP E0240 new primers
  3. Ctrl 1kb old primers
  4. Ctrl 1 kb new primers

3. Prepare culture dishes with Ampicillin, following recipe of April 20th.

4. Run gel to verify PCR to amplify GFP E0240.

PCR GFPE0240 30Jun2010.JPG

Lanes: 1) GFP E0240 old primers; 2) GFP E0240 new primers; 3) Ctrl 1kb old primers; 4) Ctrl 1 kb new primers; 5) Ladder.

5. Purify PCR product of the GFP E0240 and run another gel to verify the PCR, this because last gel was very weird ;( Purification was done with the High Pure PCR Product Purification Kit Roche. Lane 1: Ladder, Lanes 2-3: Papollo’s samples; Lanes 4-8: PCR products, PCR failed!!

Purified PCR product GFP E0240 30Jun2010.JPG