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Week16 9/26/10-10/2/10
From 2010.igem.org
Week16 Highlights
We continued work with Cp-LacPi-GFP constructs, but failed to get Cp-LacPi parts based on our gels. We also realized that our pMAL primers were incorrect so we placed an order for new primers. We completed site directed mutagenesis of CHS3.
9/26/10
Upon taking out the cultures, Kevin realized that he and Timi had put the wrong antibiotic into the incubations last night. He came back later in the day to reinoculate, but this time with Kan antibiotic!
Ran gels for the digestions --> no band was less than 1000 bp (Cp-LacPi1 parts should be ~90bp and Cp-LacPi2 parts should be ~235bp)
Is it possible that the digestions could have failed? Maybe you accidentally enabled heated lid?
The files are cplacpiscreengel1(9262010) and cplacpiscreengel2(9262010), cplacpiscreengel2overexposed(9262010) if you want to take a look. (REMOVE THIS LATER)
The bands in the gel were also a little wavy. According to Professor Mordacq, this can happen when the agarose is not properly melted all the way.
Innoculated the CP-LacPi-GFP parts again.
9/27/10
Inserted LacP-RBS into Tet plasmid --> transformed into ChiA knockouts
Kevin went in between classes to spin down the incubations from last night.
Timi picked up from there and miniprepped the samples and then digested them.
9/28/10
pMAL - realized primers are bad --> reordering correct ones
Timi went in early to do 20 uL digests of the CP-LacPI assemblies. BUT she forgot to put in restriction enzymes. FAIL.
Tried Cp-LacPi digestions using more DNA --> still too difficult to visualize the small inserts
- This could be several things (1) We just don't have any positive inserts -- which is a possibility given several unexplainable bands above 1000 bp. (2) We don't have enough DNA in the gels to properly visualize it. (3) Something with the gels/apparatus/procedure itself.
9/29/10
Site-directed mutagenesis: Amplification and DpnI digestion completed.
Ragan had some trouble with the TBE buffer. We are looking into what went wrong with it. Sean?
9/30/10
Transformation for Site-Directed mutagenesis completed.
At the lab meeting this morning, Timi discovered that Kevin had been looking for an incorrectly-sized band in the gels. They went back through the photos of gels later in the day, but were inconclusive. They are going to back-track a little bit to figure out if any of the colonies that they had picked were actually positive.
10/1/10
The transformation resulted in a good number of colonies for site-directed mutagenesis. 10 liquid cultures were made.
Timi went in early to pour a big and small gel. BUT she forgot to put in the combs. FAIL.
Kevin repoured the gels and ran the digested DNA on it. However, he forgot to run the digested parts on it as well, as the advisors had suggested.
10/2/10
Timi and Kevin put in 15 overnight inculations of 31-GFP, 32-GFP and 21-GFP.
