Team:BYU Provo/Notebook/Cholera - Enzyme/October/Period2/Dailylog

10/15/13

We ran a gel for the colony PCR products we started last night and colony 5 showed a bright band right around 1100 bp's which is the right size for DspB. We then started a 5 ml overnight in LB-Amp from its corresponding streak on the plate.

10/16/13

We ran plasmid purification on our DspB Colony E from the O/N we started yesterday. We then transformed the plasmid into BL21, which is the E. coli strain that we use for expression and purification. The transformation product was plated on LB+AMP and left in the 37° incubator overnight. All procedures were performed according to the protocols listed on our protocols page.

We also made 500 mL of LB in preparation for growing up our BL21 with DspB. The plasmid specific antibiotic, AMP, will be added tomorrow after the LB is autoclaved and ready for use.

10/17/13

We started a 5 mL overnight of our DspB in BL21 from the plate on 10/16. We also added the AMP to our autoclaved LB. Now we just have to finish waiting for our BL21 to grow up.

10/18/13

We inoculated 500 mL LB+AMP with the 5 mL overnight from yesterday and allowed it to grow for three hours from 4 am to 7 am. At 7 am we added 500 uL IPTG to induce expression of our protein and continued to grow it for another five hours from 7 am to 12 pm. At 12 pm we pelleted the bacteria and placed it in the -80°. We will purify this protein product on Monday.

10/21/13

We purified both our AmyA and DspB protein products using the His-tag purification protocol listed on the protocols page. The protein products were then run on a protein gel and [put results]