From 2009.igem.org
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| - | Descriptive Title of What You’re Doing
| + | Transformation of <i>luxCDABE</i> into pSB1AK3 |
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| - | WIKI CODING HERE
| + | * Attempted to transformed <i>luxCDABE</i> into pSB1AK3 and plated on AK plates at 37 degrees Celsius overnight. |
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Revision as of 05:46, 10 September 2009
University of Calgary
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CAROL
Transformation of luxCDABE into pSB1AK3
- Attempted to transformed luxCDABE into pSB1AK3 and plated on AK plates at 37 degrees Celsius overnight.
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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
Phosphotase, Ligation and Transformation of LuxPQ into BBK vector
Purpose: to construct and transform the LuxPQ insert and psB1AC3 insert in order to standardize the genetic part for future construction. The products from the overnight digest (July 1) underwent phosphotase treatment and subsequent ligation and transformation into TOP10 competent cells. The cells were then plated on LB+chlor plates.
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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
Research in Antifreeze Activity
We want to look at AFP, which has a very different structure in different organisms. One that we would be able to work with best (or mo
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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 NotI restriction digest
Purpose: To further verify if the J13002 + LuxOD47A + B0015 was successfully biobricked into the colonies.
Materials and methods:
These were done in accordance with the procedure outlined on the protocol page.
Results:
These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today.
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