From 2009.igem.org
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| | * Reverse primer [10 uM] (15 uL) | | * Reverse primer [10 uM] (15 uL) |
| | * Platinum Pfx DNA polymerase [2.5 units] (10 uL) | | * Platinum Pfx DNA polymerase [2.5 units] (10 uL) |
| - | Autoclaved distilled HOH (577.5 uL) | + | * Autoclaved distilled HOH (577.5 uL) |
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| - | TOTAL: 735 uL | + | * TOTAL: 735 uL |
| - | PCR reaction volume for Master Mix: 49 uL | + | * PCR reaction volume for Master Mix: 49 uL |
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| | PCR steps: | | PCR steps: |
Revision as of 22:45, 20 August 2009
University of Calgary
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CAROL
Descriptive Title of What You’re Doing
WIKI CODING HERE
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CHINMOYEE
CLASS
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EMILY
Descriptive Title of What You're Doing
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FAHD
Descriptive Title of What You're Doing
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IMAN
Descriptive Title of What You're Doing
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JAMIE
Descriptive Title of What You're Doing
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JEREMY
Preparation of TOP10 Competent Cells
In order to replicate plasmid and ensure successful transformation in the summer, today’s work was devoted to making TOP10 competent cells.
To test whether the cells were competent, pBluescript was used as a positive control, and grown on LB-Ampicillin plates. Colonies were seen the next day, verifying that the cells were made competent.
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KATIE
Improving Usability
I have found a decent way to ask questions and allow users to answer in a better way then entering things into chat. By using the llDialog function we can also communicate on negative channels, which is not possible in the chat window and helps to divide communication happening between people and between people and objects or objects and other objects.
So, I will be making everything entered in chat for the PCR machine into dialog prompt where avatars may pick from specific choices. This also gets rid of the risk of spelling errors and increases usability.
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KEVIN
Modelling papers
In order to characterize our system to the best of our ability, we have chosen the part F2620 as our role model for characterization. I have looked into and read papers regarding the rate of reactions, such as the rate of the de-phosphorylation of LuxO.
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MANDY
Descriptive Title of What You're Doing
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PATRICK
Descriptive Title of What You're Doing
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PRIMA
Descriptive Title of What You're Doing
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STEFAN
Descriptive Title of What You're Doing
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VICKI
Gradient PCR for biobrick cloning of LuxOD47A pieces
Note: I have switched with Jeremy: I will now be dealing with LuxOD47A and he will be handling LuxPQ
Purpose: To form linear copies of LuxOD47A (currently inside TOPO T/A) with BioBrick ends.
Materials and methods:
- Template: LuxOD47A in TOPO T/A [40 ng/uL]
- Forward primer: LuxOD47A BBk forward primer (Tm = 60 degrees C)
- Reverse primer: LuxOD47A BBk reverse primer (Tm = 60 degrees C)
Master Mix contents (for 15 tubes)
- 10X Pfx Amplification buffer (75 uL)
- 10mM dNTPs mixture (15 uL)
- 50mM MgSO4 (22.5 uL)
- Forward primer [10 uM] (15 uL)
- Reverse primer [10 uM] (15 uL)
- Platinum Pfx DNA polymerase [2.5 units] (10 uL)
- Autoclaved distilled HOH (577.5 uL)
- TOTAL: 735 uL
- PCR reaction volume for Master Mix: 49 uL
PCR steps:
- Denaturation: 94 degrees C, 5 minutes
- Amplification: 36 cycles of {
- Denaturation (94 degrees C, 15 seconds);
- Annealing (58-66 degrees C, 30 seconds);
- Extension (68 degrees C, 1.5 minutes)}
- Final extension: 68 degrees C, 15 minutes
- Hold temperature: 4 degrees C
12 tubes were prepared to span the gradient, each of which contained 1 uL of template DNA and 49 uL master mix. A negative control with ddHOH in lieu of template DNA was also prepared.
Results:
The gradient PCR products were visualised on a 1% agarose gel, as shown below. The right-most lane is the negative control, although it can’t really be distinguished in the results. This doesn’t seem to have worked, so it will be repeated later.
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