IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-20

To-do

• Reinoculate the Hetmann Six cultures
• Run and image gels of the colony PCR we did last night
• Send CY013 and CY014 to Genewiz again
• Transform TP (the original Topo + KaiABC plasmid) to grow up more

PCR to extract the KaiABC sequence from Cyanobacteria; the gel ran incorrectly and there was a bubble inside

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PCR to extract the KaiABC sequence from Cyanobacteria, without the bubble

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PCR to extract the KaiABC sequence from Cyanobacteria

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This is an image of a 1kb+ ladder, from the Kafatos webpage

Nick's tips for gels

• When microwaving, be certain that the broth does not boil for too long, because this will evaporate the water and the % of agarose will be higher for the gel
  • Take the flask out when this happens and gently swirl
• Make sure that the comb and the trays are clean
• Let the flask cool to a temperature comfortable to hold in your palm
• Pour gently to avoid bubbles
• When there are bubbles in the gel, use the larger end of a pipet tip to scoop the bubble out
• Cover the gel with foil to avoid bugs

Retransformation of our first Topo + KaiABC plasmid

I (Jeff) retransformed our first miniprepped Topo + KaiABC plasmid into TOP10 competent cells, since we didn't have enough plasmid to use for sequencing (Genwqiz didn't receive our order modification in time).

Since most of the miniprepped DNA was already succesfully ligated with Topo, I mixed a very small amount (2 µL of 50x dilution) with 50 µL of competent cells. I also mixed 10 µL competent cells with 2 µL H2O for a negative control. I spread 5 plates overnight: 4 experimental and 1 negative control.