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Team:Alberta/Notebook/TransformingCells
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| - | <img src="https://static.igem.org/mediawiki/2010/8/8b/TransformingCellsMap.jpg"> | + | <img src="https://static.igem.org/mediawiki/2010/8/8b/TransformingCellsMap.jpg" border="0" usemap="#Notebook_Top"> |
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| + | <area shape="rect" coords="0, 0, 171, 167" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook/TransformingCells#TSS_Transformation_Protocol"> | ||
| + | <area shape="rect" coords="277, 0, 466, 167" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook/TransformingCells#Rubidium_chloride_competent_cells_and_lyophilization"> | ||
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Transformation typically requires expensive equipment and storing competent cells is difficult for many high schools who do not have -80<sup>o</sup>C freezers. | Transformation typically requires expensive equipment and storing competent cells is difficult for many high schools who do not have -80<sup>o</sup>C freezers. | ||
We tried a few methods of transforming cells and making them competent to attempt to solve this problem in our kit. | We tried a few methods of transforming cells and making them competent to attempt to solve this problem in our kit. | ||
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==TSS Transformation Protocol== | ==TSS Transformation Protocol== | ||
| - | The TSS transformation was tested. The procedure is listed | + | The TSS transformation protocol was tested. The procedure is listed <html><a href="http://www.pnas.org/content/86/7/2172.abstract">here</a></html>. |
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| - | http://www.pnas.org/content/86/7/2172.abstract | + | |
*Acquisition of reagents- June 3, 2010 | *Acquisition of reagents- June 3, 2010 | ||
*Preparation of buffer - June 7, 2010 | *Preparation of buffer - June 7, 2010 | ||
*Cell preparation - June 7, 2010 | *Cell preparation - June 7, 2010 | ||
| - | * | + | *Protocol trial - June 10, 2010 |
| + | *Assessment of efficiency - June 11, 2010 | ||
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| + | Results: | ||
| + | Transformation with pSB1C3 gave a transformation efficiency of 4.8*10^7 colonies/μL. All colonies on the plate were strongly pink, as expected. Preparation of the cells took only 120 minutes, with a single transformation taking 90 minutes. On the other hand, a typical stock Inoue transformation cells takes a full day of work, with a single transformation taking about 120 minutes. | ||
==Rubidium chloride competent cells and lyophilization== | ==Rubidium chloride competent cells and lyophilization== | ||
| - | This method was adapted from a | + | This method was adapted from a competent cell protocol found <html><a href="http://www.unc.edu/depts/marzluff/protocols/rubidium_competent_cells.pdf" >here</a></html>, which was suggested to us by former iGEM team member Justin Fedor. A small modification to include 200mM of sucrose in each of the transformation buffers. The cells were then lyophilized overnight at 4°C on a FTS Systems Dura-Dry Lyophilizer. |
*Preparation of transformation buffer - July 6, 2010 | *Preparation of transformation buffer - July 6, 2010 | ||
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*Lyophilization - August 2, 2010 | *Lyophilization - August 2, 2010 | ||
*Transformation - August 5, 2010 | *Transformation - August 5, 2010 | ||
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| + | Results: | ||
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| + | Lyophilization proved to greater impair transformation efficiency. The determined transformation efficiency was found to be a 2*10^-1 colonies/μg. | ||
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{{Team:Alberta/endMainContent}} | {{Team:Alberta/endMainContent}} | ||
Latest revision as of 03:02, 28 October 2010
Transforming Cells
Project Timeline: Click on an image to see more information
Transformation typically requires expensive equipment and storing competent cells is difficult for many high schools who do not have -80oC freezers. We tried a few methods of transforming cells and making them competent to attempt to solve this problem in our kit.
TSS Transformation Protocol
The TSS transformation protocol was tested. The procedure is listed here.
- Acquisition of reagents- June 3, 2010
- Preparation of buffer - June 7, 2010
- Cell preparation - June 7, 2010
- Protocol trial - June 10, 2010
- Assessment of efficiency - June 11, 2010
Results: Transformation with pSB1C3 gave a transformation efficiency of 4.8*10^7 colonies/μL. All colonies on the plate were strongly pink, as expected. Preparation of the cells took only 120 minutes, with a single transformation taking 90 minutes. On the other hand, a typical stock Inoue transformation cells takes a full day of work, with a single transformation taking about 120 minutes.
Rubidium chloride competent cells and lyophilization
This method was adapted from a competent cell protocol found here, which was suggested to us by former iGEM team member Justin Fedor. A small modification to include 200mM of sucrose in each of the transformation buffers. The cells were then lyophilized overnight at 4°C on a FTS Systems Dura-Dry Lyophilizer.
- Preparation of transformation buffer - July 6, 2010
- Preparation of cells - July 10, 2010
- Preparing competent cells - July 11, 2010
- Lyophilization - July 13, 2010
- Transformation of FD cells - July 14, 2010
- Preparation of cells 2 - July 30, 2010
- Lyophilization - August 2, 2010
- Transformation - August 5, 2010
Results:
Lyophilization proved to greater impair transformation efficiency. The determined transformation efficiency was found to be a 2*10^-1 colonies/μg.
