Team:Yale/Our Project/Notebook/Week 6

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iGEM Yale

Monday 7/12--Copper growth assay of pSB74 transformants & continued ligation work

Measured the OD of the overnight LE392 cultures and found that the LE392 containing pSB74 had an OD of 0.58 while the untransformed culture had an OD of 0.87. Wanted to let the transformed culture grow a while with IPTG prior to beginning of assay (to allow time for protein expression), but didn't want either culture to overgrow in the mean time, so diluted each as follows:
To 1 mL of the untransformed solution added 4 mL of plain LB, while to 3 mL of the transformed LE392 added 2 mL of LB with Amp and 10 uL of IPTG, bringing the IPTG concentration to the desired 2 mM. Let diluted cultures grow another hour at 37˚C to reach mid-log phase.
Following the second growth period, measured the OD of the untransformed culture as 0.75 while the OD of the pSB74 transformants was 0.88, but it was diluted to 0.75.
Finally the solutions were loaded into the 96-well plate as follows, with each row having 12 wells and copper concentration decreasing left to right as before.

Miniprep and sequencing of attempt #4 transformants

Using the vacuum manifold protocol miniprepped the cultures grown up from the sixteen colonies (#9-#24) that resulted from the 4th ligation attempt.
Nanodropped three to get a sense of concentration and found that the average was 65.3 ng/uL
Prepared all sixteen samples for sequencing, taking 4 uL of each miniprep, 2 uL of 4 uM VF2 primer, and 12 uL of water.

Digest of miniprepped attempt #4 products

Digested each of the ligation products with EcoRI to linearize them prior to running them on a gel and determining their size. Each digestion reaction was composed of 5 uL of EcoRI buffer, 0.5 uL 100x BSA, 15 uL of plasmid DNA, 3.6 uL EcoRI, and 25.9 uL of water and was run overnight in the 37˚C incubator.

Colony PCR of ligation attempt #4 transformants

Ran colony PCR for each of the sixteen transformants, relying on the index plates as a cell source. Spotted cells in 20 uL of water to lyse them, then added 1 uL of the resulting solution to a PCR tube. To each tube also added 2 uL of VF2 primer (10 mM), 2 uL of VR primer (10 mM), 10.8 uL water, 4 uL 5x Phusion Buffer, 0.6 uL of DMSO, 0.4 uL dNTPs, and 0.2 uL of Phusion polymerase. Ran the reactions on the "VR" thermocycler protocol

Wetlab work for this day is also recorded on pages 59 and 62-64 of the hard copy lab notebook.

Tuesday 7/13--Transformant Copper growth assay, ligation attempt #4 results, and copper removal assay prep