Team:WashU/Week10

From 2012.igem.org

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Failed PCR in morning
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We did a PCR, but failed in the morning.
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Digestion of Z construct and tet-resistant plasmid with E and S. Run on gel
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Meanwhile, we executed digestion of Z construct and tet-resistant plasmid with E and S. Afterward, we ran the digested plasmid on gel electrophoresis.
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PCR using KlenTaq - worked [PICTURE]
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Then, we did a PCR on the digest plasmid using KlenTaq and we obtained the gel using UV-Vis. Please see the picture below.
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Transformed GC5 cells with Z-construct and U
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Plated cells to check tomorrow
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Furthermore, we have transformed GC5 cells with Z-construct and U Plated cells. We will check those tomorrow.
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<u>Tuesday, July 21</u>
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<u>Tuesday, July 31</u>
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Colony PCR on TOPO  
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Today, we did Colony PCR on TOPO and ligation of Z construct to T plasmid.
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Ligation of Z construct to T plasmid
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Meanwhile, we did PCR of Z and U constructs.
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PCR of Z and U constructs
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Meanwhile, we meet with Michael and Dan and talk about how to prepare for our iGEM E competition.
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As usual, we have a meeting in Rebstock 101 and talk about our progress about the project.
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We did a double transformation of GC5 cells with zeaxanthin-producing construct and TOPO PCR #2, and we also did that transformation with a control vector.
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Double transformation of GC5 cells with zeaxanthin-producing construct and TOPO PCR #2, done with a control vector as well
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Today, we conducted a colony PCR of the TOPO cloning of ZCD with the fusion protein (without a his-tag).  Colony PCR showed a single band of approximately 1200bp for the sample of colony number two.  A successful colony PCR should yield amplification of ZCD with a 60bp upstream region, for a total of 1170bp.  The close agreement between the band size and the expected band suggests that colony number 2 had the correct orientation of the insert (gel below).  This colony will be grown up overnight and miniprepped in the morning.
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Meanwhile, we also did a colony PCR of the U-construct. We checked the ligation of U construct and colonies of Z construct.
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Today we conducted a colony PCR of the TOPO cloning of ZCD with the fusion protein (without a his-tag).  Colony PCR showed a single band of approximately 1200bp for the sample of colony number two.  A successful colony PCR should yield amplification of ZCD with a 60bp upstream region, for a total of 1170bp.  The close agreement between the band size and the expected band suggest that colony number 2 had the correct orientation of the insert (gel below).  This colony will be grown up overnight and miniprepped in the morning.  
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Double transformation of GC5 cells with the miniprepped colony and zeaxanthin producing construct. Will induce with proper amounts of arabinose and IPTG at optimal concentrations once OD600 reaches 0.4
Double transformation of GC5 cells with the miniprepped colony and zeaxanthin producing construct. Will induce with proper amounts of arabinose and IPTG at optimal concentrations once OD600 reaches 0.4
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Separated out culture to have one at 20 degrees and one at 37, will induce both of them and see what happens
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20 degrees limits inclusion bodies
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Also ran a colony PCR on three different U colonies with two controls.
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Colony PCR of U construct with a control using the Z; U constructs failed, will redesign and redo U
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Digestion [SEE PICTURE]
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After a failed ligation of the Z-construct into an alternative, compatible plasmid with a p15A ori, the parts were analyzed and a long ligation (8hrs at 16degrees) was conducted. Multiple vector/insert ratios were also used to give the best possible chance of success. The gel used to asses the quality of the digested and purifed componenets can be seen below.
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After the long ligation protocol a gel was run to analyze the results (below).  It showed the brightest band above the ladder and some Z-construct relegated with its original plasmid. These ligations will be transformed and selected for on tetracycline LB plates.
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<div align="center"><img src="https://static.igem.org/mediawiki/2012/4/4f/Ligation.jpg" width="400"/></div>
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Latest revision as of 18:55, 17 August 2012




Monday, July 30

We did a PCR, but failed in the morning.

Meanwhile, we executed digestion of Z construct and tet-resistant plasmid with E and S. Afterward, we ran the digested plasmid on gel electrophoresis.

Then, we did a PCR on the digest plasmid using KlenTaq and we obtained the gel using UV-Vis. Please see the picture below.


Furthermore, we have transformed GC5 cells with Z-construct and U Plated cells. We will check those tomorrow.


Tuesday, July 31

Today, we did Colony PCR on TOPO and ligation of Z construct to T plasmid. Meanwhile, we did PCR of Z and U constructs. Meanwhile, we meet with Michael and Dan and talk about how to prepare for our iGEM E competition.


Wednesday, August 1
As usual, we have a meeting in Rebstock 101 and talk about our progress about the project. We did a double transformation of GC5 cells with zeaxanthin-producing construct and TOPO PCR #2, and we also did that transformation with a control vector. Today, we conducted a colony PCR of the TOPO cloning of ZCD with the fusion protein (without a his-tag). Colony PCR showed a single band of approximately 1200bp for the sample of colony number two. A successful colony PCR should yield amplification of ZCD with a 60bp upstream region, for a total of 1170bp. The close agreement between the band size and the expected band suggests that colony number 2 had the correct orientation of the insert (gel below). This colony will be grown up overnight and miniprepped in the morning. Meanwhile, we also did a colony PCR of the U-construct. We checked the ligation of U construct and colonies of Z construct.


Thursday, August 2
Miniprep of colony 2 from yesterday

Double transformation of GC5 cells with the miniprepped colony and zeaxanthin producing construct. Will induce with proper amounts of arabinose and IPTG at optimal concentrations once OD600 reaches 0.4 Separated out culture to have one at 20 degrees and one at 37, will induce both of them and see what happens 20 degrees limits inclusion bodies

Also ran a colony PCR on three different U colonies with two controls.


Friday, August 3

Colony PCR of U construct with a control using the Z; U constructs failed, will redesign and redo U

Digestion [SEE PICTURE]

After a failed ligation of the Z-construct into an alternative, compatible plasmid with a p15A ori, the parts were analyzed and a long ligation (8hrs at 16degrees) was conducted. Multiple vector/insert ratios were also used to give the best possible chance of success. The gel used to asses the quality of the digested and purifed componenets can be seen below.

After the long ligation protocol a gel was run to analyze the results (below). It showed the brightest band above the ladder and some Z-construct relegated with its original plasmid. These ligations will be transformed and selected for on tetracycline LB plates.

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