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| - | WIKI CODING HERE
| + | * PCR product was purified using sigma PCR purification kit and PCR product 1-6 were pooled together as well as PCR product 7-12 was pooled together. |
| | + | * Digested the plasmid pSB1AK3 and the PCR product using both XbaI+PstI and EcoRI+PstI enzymes for 2 hours. |
| | + | * Phosphatase the plasmid and then ligated product with PCR product using Quick Ligase. The transformation was done as well into Top 10 cells, and plates were incubated at 37 degrees Celsius overnight. |
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| + | Isolating Plasmid and Modified Colony PCR of LuxOD47E BBK |
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| - | WIKI CODING HERE
| + | Today I isolated plasmid from my overnight cultures. The best concentrations that looked the most pure was BBK LuxOD47E colony 2 cut with XbaI/PstI and BBK LuxOD47E colony 4 as cut with EcoRI/PstI. |
| | + | *I then set up a modified version of my colony PCR from yesterday, using only these 2 colonies and LuxOD47E C4-8 in the pCR2.1-TOPO vector and the psB1AC3 vector as positive controls. Cycling conditions were as follows: 94ºC for m minutes, 36x (94ºC for 45s, 55ºC for 45s and 72ºC for 2 minutes), 72ºC for 10 minutes. Held at 4ºC. |
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| - | Today, I helped organize our second bake sale (Cultural Bake Sale: The Sequel) of our marketing campaign. The bake sale managed to raise more than 370 dollars. We had cool baked items such as samosas, spring rolls, muffins and lots lots and lots of other stuff. I want to thank my entire iGEM calgary team for their tremndous support and countless effort the put in for the event. | + | Today, I helped organize our second bake sale (Cultural Bake Sale: The Sequel) of our marketing campaign. The bake sale managed to raise more than 370 dollars. We had cool baked items such as samosas, spring rolls, muffins and lots lots and lots of other stuff. I want to thank my entire University of Calgary iGEM team for their tremendous support and countless effort the put in for the event. |
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| + | The Beginning of a Short Lab Hiatus to Write! |
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| - | WIKI CODING HERE
| + | Time was spent writing a paper detailing the trials and tribulations of luxOU construction to help internalize and review microbiology concepts and techniques. Next pressing update: July 6, 2009. |
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| - | Purpose: to move linear LuxPQ with BBK restriction sites into the standard psB1AC3 vector. The LuxPQ PCR product was purified by pooling 4 X 40uL of product and following the Qiaquick PCR purification protocol. This productwas then digested with XbaI and PstI, as was psB1AC3 for 2 hours at 37ºC. After phosphotase treatment and ligation, the product was transformed into TOP10 competent cells and plated on LB+chlor plates overnight. | + | Purpose: to move linear LuxPQ with BBK restriction sites into the standard psB1AC3 vector. The LuxPQ PCR product was purified by pooling 4 X 40uL of product and following the Qiaquick PCR purification protocol. This product was then digested with XbaI and PstI, as was psB1AC3 for 2 hours at 37ºC. After phosphotase treatment and ligation, the product was transformed into TOP10 competent cells and plated on LB+chlor plates overnight. |
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| | *1099bp long | | *1099bp long |
| | *They have a relatively weak TH activity of ~0.1-0.5°C, which is less than that of fish AFPs and significantly less than that of insect AFPs. | | *They have a relatively weak TH activity of ~0.1-0.5°C, which is less than that of fish AFPs and significantly less than that of insect AFPs. |
| - | *The TH activity of recombinant proteins from an E.coli expression system is weak | + | *The TH activity of recombinant proteins from an <i>E. coli</i> expression system is weak. |
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| - | Today, I followed up with almost all the companies which I was in charge of contacting and sent them the June Newsletter. I thanked all those companies who have sponsored our project and encourageed everyone to visit our wiki for details on our progress. I also attached the CTV link to every email. | + | Today, I followed up with almost all the companies which I was in charge of contacting and sent them the June Newsletter. I thanked all those companies who have sponsored our project and encouraged everyone to visit our wiki for details on our progress. I also attached the CTV link to every email. |
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| - | WIKI CODING HERE
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CAROL
PCR product Purification and Restriction Digest
- PCR product was purified using sigma PCR purification kit and PCR product 1-6 were pooled together as well as PCR product 7-12 was pooled together.
- Digested the plasmid pSB1AK3 and the PCR product using both XbaI+PstI and EcoRI+PstI enzymes for 2 hours.
- Phosphatase the plasmid and then ligated product with PCR product using Quick Ligase. The transformation was done as well into Top 10 cells, and plates were incubated at 37 degrees Celsius overnight.
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CHINMOYEE
Bioprocess Engineering
Looked into my bioprocess engineering book for useful information and different aspects to consider for the model.
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EMILY
Isolating Plasmid and Modified Colony PCR of LuxOD47E BBK
Today I isolated plasmid from my overnight cultures. The best concentrations that looked the most pure was BBK LuxOD47E colony 2 cut with XbaI/PstI and BBK LuxOD47E colony 4 as cut with EcoRI/PstI.
- I then set up a modified version of my colony PCR from yesterday, using only these 2 colonies and LuxOD47E C4-8 in the pCR2.1-TOPO vector and the psB1AC3 vector as positive controls. Cycling conditions were as follows: 94ºC for m minutes, 36x (94ºC for 45s, 55ºC for 45s and 72ºC for 2 minutes), 72ºC for 10 minutes. Held at 4ºC.
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FAHD
Bake Sale Sequel, June 30th 2009
Today, I helped organize our second bake sale (Cultural Bake Sale: The Sequel) of our marketing campaign. The bake sale managed to raise more than 370 dollars. We had cool baked items such as samosas, spring rolls, muffins and lots lots and lots of other stuff. I want to thank my entire University of Calgary iGEM team for their tremendous support and countless effort the put in for the event.
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JAMIE
The Beginning of a Short Lab Hiatus to Write!
Time was spent writing a paper detailing the trials and tribulations of luxOU construction to help internalize and review microbiology concepts and techniques. Next pressing update: July 6, 2009.
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JEREMY
Moving linear LuxPQ into BBK vector
Purpose: to move linear LuxPQ with BBK restriction sites into the standard psB1AC3 vector. The LuxPQ PCR product was purified by pooling 4 X 40uL of product and following the Qiaquick PCR purification protocol. This product was then digested with XbaI and PstI, as was psB1AC3 for 2 hours at 37ºC. After phosphotase treatment and ligation, the product was transformed into TOP10 competent cells and plated on LB+chlor plates overnight.
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KEVIN
Search for other antifreeze proteins
Carrot's antifreeze proteins were also looked into.
- 1099bp long
- They have a relatively weak TH activity of ~0.1-0.5°C, which is less than that of fish AFPs and significantly less than that of insect AFPs.
- The TH activity of recombinant proteins from an E. coli expression system is weak.
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MANDY
Meeting with Dr. Jacob for Second Life
Today, we went over to main campus to meet up with Dr. Jacob and discuss our current progress. We also continued discussion about when and how we would beta test our project, and how to make it more intuitive and educational. Katie and I looked at a Biobrick circuit assignment given to us by Thane from the beginning of the year. We began modelling possible biobricker levels based on the concepts shown in these circuits.
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PATRICK
HUD Details
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
Spent today working out some details of how the tile control system would work. Should I erase the entire screen before drawing the next one, or just let the next screen's text and buttons replace all of the current display? Decided to blank first instead of relying on the next screen to replace it all. Sending a single message to all display tiles to pre-blank had a low overhead cost, the update process looked cleaner on the HUD, and I could then skip space characters when processing the next screen's text. (No need to tell tile x to display a space character, if it has been pre-blanked).
Set up a system for parsing lists of characters into lines on the UI, so that I could easily visualize what the display would look like in the program's code. e.g., an early version of the main menu:
display += " Biobrick ";
display += " Simulator ";
display += " ";
display += " X Introduction";
display += " X Levels ";
display += " X Sandbox ";
display += " X Help ";
display += " ";
The Xes here stand in for buttons.
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PRIMA
Marketing
Today, I followed up with almost all the companies which I was in charge of contacting and sent them the June Newsletter. I thanked all those companies who have sponsored our project and encouraged everyone to visit our wiki for details on our progress. I also attached the CTV link to every email.
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VICKI
Sequencing of the LuxOD47A BBk + B0015 colonies
Based on yesterday's NotI restriction digest results, the LuxOD47A + B0015 colonies look promising. These were prepared with BBk construction primers and sent for sequencing, with positive results eventually confirmed. These are attached below.
Construction protocol: adding J13002 to LuxOD47A + B0015
Cutting with (EcoRI+SpeI for the J13002 part) and (EcoRI+XbaI for the LuxOD47A + B0015 part) restriction enzymes, Antarctic phosphatase treatment, ligation, transformation into competent TOP10 cells and plating on AK plates were all done in accordance with the procedures outlined on the protocol page. If this is done properly, the net J13002 + LuxOD47A + B0015 will be on an pSB1AK3 plasmid.
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