October 7th-October 11th

Week Three (Oct 7-11)


On the day of the seventh, we discussed melting and plating LB broth with 500 ul of ampicillin. Since this was my first time doing any sort of work with media, we went over dating, how to plate, and the process of melting the media.

  • For media: [Name of Media] [Amount of Media] [Date] [Initials]

  • Ex: LB 250 ml 10/07 J.M.

  • Keep LB broth at a heat of five and stir using heat glove to avoid boiling over

  • This time, we plated half with plain LB broth and half with LB broth and 500 ul of ampicillin.

On the eighth, we planned on putting out pgK on a gel to see bands. With this, we got successful ligation. After, we took a tube of pgRNA that we know had been cut by xbar successfully and added xbam to that.


Additionally, on the ninth, we discussed how we previously took our digested kana and dosed it with xbar and was now planning on ligating and PCR. We also discussed the actual process of PCR.

  • Step one: PCR is essentially targeted replication. The heat of our machine causes breakage, thus the hydrogen bonds holding DNA will separate into single stranded DNA.

  • Step two: Here, the DNA will continue to separate more.

  • Step three: The primers go in at a kneeling temperature and go to their locations which allows the polymerase to know where to go. The best temperature for these primers is 57°C.

  • Step four: Riding to 72°C, the polymerase has gone from one strand to two.

  • Step five: The machine will cycle back to step two and repeat the process for alloted amount of time.

Essential Notes:

  • pdCas9 has 6,700 bps.

  • Most deinos prefer to grow at anywhere between 30°C-37°C.

  • Sonora prefers 30°C.

  • E. Coli prefers 37°C.


We also ran a gel on our pgK and was confused by the results. We didn’t reach a conclusion about what went wrong.

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