Team:UMBC-Maryland/Notebook
Notebook
'''Week 1'''[[June 1st]]
First meeting of the project over summer where we ran our PCR products on a 1% agarose gel with results shown in 6_1_15PCR.jpg. Band sizes are determined from the ladder in N0467_thumb.gif.
[[June 4th]]
iGEM team members attended a meeting with a professor and was given an overview to the procedure of 3A Assembly. We also discussed team structure and the CUP1 gene construct that is needed to be synthesized as a G-block. G-blocks are then submitted to IDT for sequencing.
'''Week 2'''
[[June 8th]]
Digest products with a combination of restriction enzymes (XbaI, HindIII, EcoRI)were ran on a 2% agarose gel. Both gel results came up inconclusive.
'''Week 3'''
[[June 15th]]
Repeated digestion to troubleshoot any possible errors that may have occurred during the protocol.
'''Week 4'''
[[June 22nd]]
Repeated analysis of the digestion products on the gel. Gel results showed no bands
[[June 25th]]
Mini-prep of the metallothionein plasmid.
'''Week 5'''
[[June 29th]]
Digestion of the metallothionein plasmid with restriction enzymes (EcoRI & PstI). After further investigation, the Bulls-Eye solution was the cause of bands not appearing and we switched to Ethidium Bromide. The digestion products were then run on a gel stained with ethidium bromide. The gel depicted both digested and undigested bands that can be used to identify whether our restriction enzymes are working or not.
'''Week 6'''
[[July 7th]]
Today marks the first day we began production of our first cell line, Retriever 1(Ret1).The CUP1 transformed cells and control cells were plated and an overnight was done.
[[July 9th]]
Inoculation experiment. Six flasks of 25 mL LB broth were prepared, 3 of which will be the control and the other will be for the transformed. From Copper Sulfate, a stock solution of 0.5M Copper Solution was created and then a 0.1M Solution was created to be used. Enough volume of the 0.1M Copper Solution was added to the 6 six flasks so each of the control and transformed group will have flasks containing a concentration of [0uM], [15uM] and [25uM]. To the transformed group 9.375uL of Chloramphenicol was added. At every hour for eight hours, starting at the 0th hour, an optical density reading will be taken for each flask. Drawing out 1mL of the solution from each flask into eppendorfs, followed by measuring the optical density. Since the Laemmli Buffer was nowhere to be found,the solution was spun down to be put in the freezer for the SDS gel in the future. Another 1 mL from each flask is saved for copper measurement. Data of the time points is depicted as a table and graph.
'''Week 7'''
[[July 15th]]
Meeting with our advisor regarding current updates on the project and the availability of the flame atomic absorption spectrometer to be used for the copper measurement. An overnight for the Ret1 transformed cells was done today to repeat the experiment at higher concentrations of copper.
[[July 16th]]
Another inoculation experiment was scheduled for today, however the tube of the overnight cells broke so instead a repeat of the overnight was done.
[[July 17th]]
The second inoculation experiment. A copper concentration of [0uM], [50uM], [75uM] and [100uM] was experimented with. Data was collected in
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