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Team:Calgary/15 June 2009
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| - | + | JUNE 15, 2009 | |
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| - | + | Continuation of Plasmid Isolation | |
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| - | + | * Repeated Maxi-prep protocol and was unsuccessful. | |
| + | * Will attempt to continue on single site mutagenesis and then will continue looking at other protocols to isolate higher concentration of plasmid. | ||
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| - | + | Research : how does the Stochastic simulation for Simbiology work ? | |
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| - | + | The Stochastic simulation works with Gillespie's Algorithm . There is more than one version of Gillespie's algorithm : Direct Method , () | |
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| + | Direct Method : | ||
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| - | + | Gradient BBK Amplification of TOPO Trial Three | |
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| - | + | *See protocol on June 6th 2009 | |
| + | *PCR Products were visualized on a 1% agarose gel with 1.0 kb Generuler DNA Ladder. | ||
| + | *See gel photo below. | ||
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| + | [[Image:2009.06.18D47E_BBKVer_GradientPCR.jpg|350px]] | ||
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| + | *Lanes 1-9 contain LuxOD47E in pCR2.1-TOPO vector, lane 10 is a (clean!) negative control. | ||
| + | *Analysis: Because the negative control lane is clean (no bands) indicating that there is no contamination of my products, we will move on to moving my gene of interest (LuxOD47E) from the pCR2.1-TOPO vector into the Biobrick psBA1C3 vector. | ||
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| - | + | Marketing for June 15th 2009 | |
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| - | + | Today, I called a couple of pharmaceutical and O&G development companies. I left a voicemail for majority of them. I also started working on the June Newsletter and mailed out our sponsorship package to Paladin Labs. | |
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| - | + | Retransformation of luxOU in psB1AC3 | |
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| - | + | Tested TOP10 transformation using pBluescript and with varying temperatures/times of 37oC for 5 minutes and 42oC for 45 sec. Plated on either Cm35 (construction) or Amp100 (positive control) plates as required. | |
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| - | + | PCR Purification of LuxPQ with BBK RS; construction into BBK plasmid; transformation | |
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| - | + | 40uL of 4 gradient PCR products of luxPQ were pooled and then purified following the QIAquick PCR purification protocol. Product purity and concentration were measured using the NanoDrop Spectrophotometer. The linear luxPQ and pure psB1AK3 were then cut with the following sets of enzymes in separate tubes for construction of LuxPQ into the BBK vector: XbaI and PstI; EcoRI and PstI. This was allowed to digest for 2 hours, then underwent phosphotase and ligation treatment, and was subsequently transformed into TOP10 competent cells. These cells were then plated on LB agar + Kanamycin plates overnight at 37ºC. | |
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| - | Editing of AI-2 signalling system model on | + | Editing of AI-2 signalling system model on Symbiology. No experiments were performed. |
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| - | + | Testing Katie's PCR & Xyzzy | |
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| - | + | While testing Katie's PCR machine, I might have sort of broke it a bit. The objects with scripts in them that I dropped in them did not go away, and in addition, scripts that caused the PCR machine to glow, etc. could not be removed. Katie, who is amazing, has been developing a cleaning script that will solve this problem. | |
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| + | We noticed that Sigma Aldrich had a workable HUD for their lab activities that used a text script allowing prims to easily display and change text. I found out that it was the XYZZY script, and using the LSL wiki, I set about making a billboard that people can write on, to play with this script. However, it is really complicated and only with a LOT of guidance from Patrick was I able to make a primitive one. I've gone back to the Science Building in SL to review core concepts of scripting that I missed. | ||
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| - | + | Mrketing continued | |
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| - | + | I followed up with many oil and gas companies. I left a voice mail with all the companies today because no one was available at the office today. | |
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| + | I received my lab notebook today! I'll start filling it out ASAP. | ||
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| - | + | VICKI | |
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| - | + | Construction of LuxOD47A BBk into psB1AC3 | |
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| - | + | * Antarctic phosphatase treatment | |
| + | ** Purpose: to prevent to formation of a phospodiester bond between ccdB and psB1AC3 | ||
| + | ** Protocol: The vector (psB1AC3 + ccdB) tubes were treated in accordance with the Antarctic phosphatase protocol on the protocol page. | ||
| + | * Ligation of LuxOD47A BBk to psB1AC3 | ||
| + | ** Purpose: to ligate the insert to the vector in BioBrick form (so that all of the BioBrick sites are preserved). | ||
| + | ** Protocol: the tubes were treated in accordance with the Ligation protocol on the protocol page. | ||
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| - | + | Transformation of LuxOD47A BBk on psB1AC3 into competent TOP 10 cells | |
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| - | + | Purpose: The constructs are in a water solution, where they will be stable for about 2 weeks. If they are in competent cells with natural DNA repair mechanisms, they will be stable for much longer. To accommodate this, the constructs need to be inserted into competent cells. | |
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| + | Protocol: The transformation was conducted in accordance with the transformation protocol on the protocol page. pBluescript was also transformed into a set of cells to act as a positive control. We’re using an AC-resistant vector, so any bacteria that are properly transformed with some non-ccdB gene on the AC plasmid will survive. pBluescript does not afford AC resistance, so cells transformed with that will die. We already tested the plates to ensure that the antibiotic works, so there’s no need for a negative control. | ||
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| + | Once the transformation occurred, plates were made of the transformed cells so that distinct colonies could grow, in accordance with the culture plate protocol on the protocol page. | ||
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Latest revision as of 06:21, 20 October 2009
UNIVERSITY OF CALGARY
