Jeff:LabNotes/Microbiome/2010-11-19

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BLAST Results[edit]

Received Sanger Sequencing Results from 11-18-10

  • 7 total samples were sent to Genewiz
  • 2 failed due to priming
  • 3 did not have an insert in it. These colonies should have turned blue. Based on my experience, I think you need to wait at least 1.5 days for the XGAL to take affect. In this case, I only waited a day so the blue colors could have been very faint.
  • Of the remaining 2 samples, they both blasted 100% to unculturable bacteria as well as 98% to ecoli. This unculturable bacteria is definitely contamination. I should expect a 100% match to e coli as I had gotten in the past

Contamination Discussion

Where is it coming from? Need to find source and isolate. Since I UV everything in hood and wipe down hood with ethanol, it is unlikely that the contamination is coming from this. My best guess is during the micromanipulation and microscope use the platform is not clean. I need to clean everything thoroughly with ethanol.

Dr Zhang's/My Ideas

  • Do pWGA in tubes and see if contamination exists. This will tell us if its pre or post amplification.
  • Also, inject amplicons of specific size into wells. Use another micropipette to remove. See if any contamination after removal
  • Spike wells with gDNA to see if amplification occurs in most wells
  • Create larger well size (200-400 um) to dilute out contamination.