Team:BYU Provo/Notebook/Cholera - Enzyme/July-August/Period3/Dailylog


Cholera - Enzymes Notebook: August 1 - August 14 Daily Log

Cholera Enzyme


We ran the restriction digests out on low melt gel and set up ligations with DspB and the Pet15b vector. The ligation was left overnight at room temperature per the protocol listed on the protocols page.


We set up transformations into BL21 E. Coli for the DspB+Pet15b plasmid. The transformation was run per the protocol listed on the protocols page and was left overnight in the 37°C incubator.


We checked the transformation of our DspB+Pet15b plasmid into E. coli, but the transformation was not successful. We re-setup the colony PCR for DspB and Savinase.

We received a frozen culture of V. cholerae from Dr. Robison’s lab so that we can grow new plates and overnights. Cells are in 20% glycerol.


The DspB colony PCR didn’t work. We re-ran the PCR yesterday, so we will run the product on gel today, clean up the PCR and sequence it. We also prepped the Savinase for sequencing today since the DspB didn’t work and we don’t want to wait any longer. We also streaked a new plate of cholera with the culture that we received from Dr. Robison’s lab. New plate was placed in the 37° incubator.


Phusion PCR of Savinase and DspB. We ran two samples of each with a control:

  1. DspB
  2. DspB
  3. DspB Control
  4. Savinase
  5. Savinase
  6. Savinase Control


We ran DspB and Savinase on gel but the results were not promising. There were two very faint bands for DspB, but not enough for us to use. Savinase did not have any bands showing at all. We then reran the Phusion PCR for our samples following the protocol listed in the Grose lab protocol packet.

Our samples were:

  1. DspB
  2. DspB Control
  3. Savinase
  4. Savinase Control

We also reseeded new cholera overnights by adding 4 mL of SLB and 50 uL of our previous cholera overnights.


We set up PCR again for DspB and Savinase. The PCR tubes were labeled:

  1. DspB
  2. Savinase


We ran the PCR products from yesterday on gel, but nothing showed, not even the ladder. This indicates that when the gel was prepared, the ethidium bromide was not added as it was supposed to be.

We made a new gel and re-ran our PCR products:

  1. Ladder
  2. DspB
  3. DspB Control
  4. Savinase
  5. Savinase Control

There was a faint band for the DspB, but nothing for Savinase. We will try to move forward with our DspB, but may need to restreak the Bacillis subtilis for our Savinase enzyme.