Week Four - 8 July to 14 July


Brian transformed an ampicillin resistant plasmid into the top10 competent cells from last week in order to test their effectiveness
Yuan is making another batch of top10 competent cells in case the previous batch is not competent enough


9:00 am to 11:00:
Mathworks matlab seminar on using matlab more efficiently when analyzing data. Was very helpful for our modeling team (Mike and Grant and Brian)

9:30 am to 11:00:
Rainen and other sponsors held a breakfast discussion section meeting. Lajja and Sarah went to discuss product sponsorship.

Yuan worked in the lab continuing to research genomes and coding regions that were needed for all of our parts for pcr and synthesis.

Brian tested the efficiency of the transformed e. coli competent cells as outlined in the protocol section:
Concluded that the cells were 10^5 efficiency, which while slightly too low for the purposes of our final design, would work well for transforming supercoiled parts from the registry
Yuan started making more competent cells at 10^6 efficiency


Continued searching for the coding regions of the phytases and the entire genomes of the organisms they came from (for pcr)
Also continued searching for the coding regions of the parts and the genomes of the organisms they came from (for pcr)

Jennifer came to the lab and taught us about how pcr works and how to assemble the primers for pcr. Specifically, there is a calculator online that helped with forming the primers for pcr:

The heuristic rules for primers were as follows:

  • Avoid primers that are complementary to one another.
  • Avoid primers that have internal complementarities—it can lead to hairpin formation.
  • Be sure that the 3' ends of the primers are exactly complementary to the template strand.
  • Primers that end in C or G are most efficient.
  • Primers should be about 50% G+C in content.
  • Tm for primers should be similar to one another within 3 degrees C.
  • Primers should have a Tm (melting temperature) between 55-75 degrees C.

Mike came to the lab and helped us learn more about the different assembly methods, specifically the standard method and the 3A method. We now have a plan for cutting and pasting our parts together into the backbone plasmid using the restriction enzymes.

We are waiting on the advisors to approve our concept plan before initiating buying the supplies.


Practice transformations:

Added 10 microliters of nuclease free water to the following wells
Waited 5 minutes.

Lajja and Sarah: GFP(plate 2 well 8, A)
Brian and Yuan: RFP (plate 2 well 6, O)
Grant and Mike: CFP (plate 1 well 24, E)

Put 25 microliters of competent cells into a culture tube.

Take the DNA out of the well, push out the DNA in the tip into the culture tube of cells, release the tip into the culture tube, wait 30 minutes

Water bath 42 seconds

Incubate 1 hour

Put 10, 50,100 microliters of cells. 100 of SOB in first one (10microliter)
Then put cells in incubator to culture overnight