From 2009.igem.org
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| - | Descriptive Title of What You're Doing
| + | Repeat of colony PCR of J13002 + LuxOD47A + B0015 |
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| - | WIKI CODING HERE
| + | The purpose and materials/methods are the same as they were yesterday. This time, only 4 colonies were tested, along with the same size controls as yesterday. |
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| | + | Results: |
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| | + | The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls. |
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| | + | Plasmid isolation and restriction digest |
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| | + | Purpose: To further verify if the J13002 + LuxOD47A + B0015 was successfully biobricked into the colonies. |
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| | + | Materials and methods: |
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Revision as of 23:52, 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
Descriptive Title of What You're Doing
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EMILY
Descriptive Title of What You're Doing
- Protocol: Used GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH2O.
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| psB1AC3 1 | 1.96 | 3.48 | 76.9
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| psB1AC3 2 | 1.93 | 2.29 | 98.1
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| psB1AC3 3 | 1.87 | 1.88 | 48.5
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| psB1AC3 4 | 1.90 | 2.11 | 118.9
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| psB1AK3 1 | 2.05 | 2.42 | 134.4
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| psB1AK3 2 | 1.95 | 2.23 | 55.6
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| psB1AK3 3 | 1.91 | 2.22 | 97.9
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| psB1AK3 4 | 2.03 | 1.92 | 44.5
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Verification digest to verify the presence of restriction sites in the psB1AC3, psB1AK3 vectors as well as in our TOPO TA vector with LuxOD47E.
5 reactions for psB1AC3 vector, 5 reactions for psB1AK3 vector, 1 reaction for gene of interest (LuxOD47E in pCR2.1-TOPO vector).
BBK Vector Reaction List
Reaction 1- Not1 + React 3 Buffer
Reaction 2- EcoRI + SpeI + React Buffer 4
Reaction 3- XbaI + PstI + React 2 Buffer
Reaction 4- EcoRI + PstI + React 2 Buffer
Reaction 5- XbaI + SpeI + React 4 Buffer
BBK vector tube preparation
8 uL Plasmid (psB1AC3 or psB1AK3)
2 uL appropriate React Buffer
1 uL of each appropriate restrcition enzyme
ddH20 up to 20 uL
TOPO vector tube preparation
10 uL DNA (LuxOD47E in pCR2.1- TOPO vector)
7 uL ddH20
2 uL React 3 Buffer
1 uL NotI restriction enzymes
Left to digest overnight in waterbath at 37°C.
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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
Descriptive Title of What You're Doing
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KATIE
Descriptive Title of What You're Doing
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KEVIN
Continuation of the research in Antifreeze proteins
Usefulness of the signalling system in antifreeze activity
For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue.
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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
Repeat of colony PCR of J13002 + LuxOD47A + B0015
The purpose and materials/methods are the same as they were yesterday. This time, only 4 colonies were tested, along with the same size controls as yesterday.
Results:
The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls.
Plasmid isolation and restriction digest
Purpose: To further verify if the J13002 + LuxOD47A + B0015 was successfully biobricked into the colonies.
Materials and methods:
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