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From 2014.igem.org

BYU 2014 Notebook Edit September October

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Week of September 6th

September 3, 2014

--CS-- Today I checked the sequencing results from all of last week's sequencing. The samples that I submitted for nirS (3-5) and norC (3-2 and 3-4) all turned out great! The results showed that the promoters were inserted into the plasmids upstream of the gene sequences, just as we wanted them. I also checked the norB mutagenesis results. It appears that they did not work properly, so we'll have to try those all over again. The PCR hasn't been working properly, so I think I should try starting over from there instead of transforming and everything all over again. Julie and I also talked and decided that Julie would take over with constructing the ginormous plasmid containing all four of the denitrification genes since she is pretty much done with her antibiotics stuff now.

Week of September 13th

September 8, 2014

--CS-- Today I reviewed where everything is at right now with the denitrification project. I transformed 2 μl and 4 μl of the DpnI-digested nosZ into DH5α, letting it incubate at 37°C for 1.5 hours, and then plated it onto LB+Cam plates, putting them in the old 37°C incubator overnight.

September 9, 2014

--CS-- The nosZ plates that I grew up overnight didn't have any colonies, so I will have to start the mutagenesis reaction all over since it hasn't worked for the past few times. Today I did a new colony PCR reaction for the norB mutagenesis reactions since the past few times haven't really worked.

September 10, 2014

--CS-- Today I ran an analytical gel of the norB PCR products. My gel turned out great this time around! Here is the image:

I will be able to sequence a few of these this time and hopefully will be able to tell if the mutagenesis actually worked or not since all of the other tests failed to have very reliable results.

I also went back to old plates and picked a colony for nosZ from before mutagenesis but after successful cloning to use for plasmid preps prior to redoing the mutagenesis reaction. I also whipped up a new P. aeruginosa PAO1 stock plate just in case we need that anymore the rest of the semester since the stock plates we have are getting old. I also contacted Brother Lee about getting some Durham tubes made up.