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Team:BYU Provo/Notebook/Cholera - Enzyme/May-June/Period2/Dailylog
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Revision as of 16:48, 26 September 2013
| Cholera - Enzymes Notebook: May 15 - May 31 Daily Log
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5/15/13 We ran phusion PCR samples on regular agarose gel. See products results below [IMAGE] We need to re-setup the PCR products as there was no visible band for either the AmyA or Savinase proteins on our gel.
5/16/13 We reran phusion PCR for AmyA and Savinase. In addition, we also re-boiled the DNA template for Subtilisin savinase. In the PCR master mix we used a GC base thermobuffer in place of the normal 5X Phusion buffer. We hope this will help the PCR reaction for our enzymes.
5/17/13 We ran our phusion PCR products on agarose gel. See the results below. [IMAGE] Row 1: AmyA control The AmyA products worked. There was no visible band for the Savinase products. It seems like we are not getting adequate DNA from our lysed Bacillus Subtilis cells. We need to determine a new boiling method to better isolate the Savinase DNA in preparation for the PCR.
5/20/13 We set up our first 96-well plate for the biofilm assay. We want to determine the best incubation time for the plate to have sufficient biofilm growth for us to run the assay once the enzymes are ready. For our initial determination, we set up the plate as shown in the tables below. The plate was placed in the 30 °C incubator as our biofilm shows the strongest growth at this temperature.
5/21/13 We added 50 ul of media to their respective wells to prevent evaporation of the samples. The media was carefully added by dispensing the media along the side of each well.
5/22/13 Today we again added 50 ul of each media to ensure biofilm growth restoration. The media was carefully replaced by dispensing the media along the side of each well. Future plans:
5/24/13 We prepared 500 mL of new SLB media following the recipe listed on 4/3/13. The exact measurements for salt added to the synthetic sea water were:
5/29/13 We set up AmyA cloning with plasmid pIG 86 and pet 15b vector (pJG 648) for cloning. We set up restriction digests with the following protocol, using BamH1 and Nde1 as our restriction enzymes:
We left the restriction digests overnight in the 37° water bath. Today we received the Dispersin B plasmid (iGem part BBa_K802001) that we had requested from the Lyon-INSA iGem Team, who were working with this part last year. The sample was inadequately packed in an eppendorf inside of a plastic test tube. The test tube was shattered and the eppendorf was cracked along the side. We performed an E. coli transformation for Dispersin B and added 10ul to the eppendorf to attempt to rescue the DNA sample. We then transferred this into DH5-alpha cells and followed E. coli TFN protocol. TFN was plated on LB-AMP and left in 37° incubator overnight.
5/30/13 TFN for DspB was unsuccessful; we were unable to recover any of the sample. We requested another sample of iGem part BBa_K802001 from the Lyons-INSA iGem team, hopefully this sample will arrive intact.
5/31/13 Ran pet15b vector and AmyA restriction digests on low melt gel. Results shown below: [IMAGE] There were no digest products for AmyA. We need to reset up the digest and recheck the restriction enzymes.
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