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Team:Calgary/28 May 2009
From 2009.igem.org
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| - | + | Gel electrophoresis of PCR and restriction digest products from yesterday | |
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<div class="desc"> | <div class="desc"> | ||
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| - | + | Purpose: To analyse the PCR and restriction digest products from yesterday by running them on the gel. This will show: | |
| + | - Whether or not the products are free of DNA contamination | ||
| + | - Whether or not they actually contain LuxPQ BBk (or LuxPQ TOPO) – note that sequencing is the only way to confirm this, but this will help us eliminate products that clearly do not contain the gene of interest | ||
| + | |||
| + | Materials and methods: | ||
| + | 1. Preparation. | ||
| + | a. Salvaging the restriction digests, which were hastily left overnight at room temperature and with no orange dye to stop the reaction. This was addressed today, when 4uL of orange dye were introduced to the 20 uL of restriction digest tube contents to deactivate the NotI restriction enzymes. 16 uL of ddHOH were added to bring the total tube volume up to 40 uL | ||
| + | b. Introducing a negative control. Stock LuxPQ BBk (of the uncut variety) will produce a distinct band on the gel around 7500 bp (accounting for the psB1AC3 plasmid and the LuxPQ gene), with fluctuations in levels based on the degree of coiling. Accordingly, if the NotI restriction digests did not actually digest, the digest lanes will resemble this control. This tube included 2uL of DNA, 1 uL of dye and 7 uL ddHOH | ||
| + | c. PCR products to evaluate specificity. If there are significant genetic mutations in the BBk sample, the primers will either not anneal at all (resulting in no PCR product) or anneal but give a product that is of a different size (if the mutations are beyond the primer domain). | ||
| + | 2. Gel set-up | ||
| + | a. A 0.7% agarose gel was made. 10 uL of the aforementioned cocktails were loaded into each well, with the well key included in the results picture. Voltage was set at 90V and left to run for 50 minutes. A GenRuler 1 kb Plus DNA Ladder was run in parallel to provide size calibrations for our results. | ||
| + | |||
| + | Results: | ||
| + | (insert May 28 gel picture here) | ||
| + | |||
<html> | <html> | ||
Revision as of 22:12, 31 July 2009
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CAROL
Modeling Readings Continued
LuxCDABE-M-F:CCATTAATGAATTGCCGGATAATCTGGATTTTGAAGGCC LuxCDABE-M-R:GGCCTTCAAAATCCAGATTATCCGGCAATTCATTAATGG
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CHINMOYEE
Descriptive Title of What You're Doing
WIKI CODING HERE
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EMILY
Sequencing of LuxOD47E in psB1AC3
File:2009.05.28.D47E BBkVer RD+PCR.tif
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FAHD
Descriptive Title of What You're Doing
WIKI CODING HERE
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IMAN
Descriptive Title of What You're Doing
WIKI CODING HERE
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JAMIE
Descriptive Title of What You're Doing
WIKI CODING HERE
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JEREMY
Descriptive Title of What You're Doing
WIKI CODING HERE
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KATIE
Descriptive Title of What You're Doing
WIKI CODING HERE
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KEVIN
Matlab and symbiology
Familiarizing with the matlab program provided by MathWorks, and its biological tool called Symbiology.
No experiments were done on this day.
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MANDY
Descriptive Title of What You're Doing
WIKI CODING HERE
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PATRICK
Descriptive Title of What You're Doing
WIKI CODING HERE
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PRIMA
Descriptive Title of What You're Doing
WIKI CODING HERE
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STEFAN
Descriptive Title of What You're Doing
WIKI CODING HERE
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VICKI
Gel electrophoresis of PCR and restriction digest products from yesterday
Purpose: To analyse the PCR and restriction digest products from yesterday by running them on the gel. This will show:
- Whether or not the products are free of DNA contamination
- Whether or not they actually contain LuxPQ BBk (or LuxPQ TOPO) – note that sequencing is the only way to confirm this, but this will help us eliminate products that clearly do not contain the gene of interest
Materials and methods: 1. Preparation. a. Salvaging the restriction digests, which were hastily left overnight at room temperature and with no orange dye to stop the reaction. This was addressed today, when 4uL of orange dye were introduced to the 20 uL of restriction digest tube contents to deactivate the NotI restriction enzymes. 16 uL of ddHOH were added to bring the total tube volume up to 40 uL b. Introducing a negative control. Stock LuxPQ BBk (of the uncut variety) will produce a distinct band on the gel around 7500 bp (accounting for the psB1AC3 plasmid and the LuxPQ gene), with fluctuations in levels based on the degree of coiling. Accordingly, if the NotI restriction digests did not actually digest, the digest lanes will resemble this control. This tube included 2uL of DNA, 1 uL of dye and 7 uL ddHOH c. PCR products to evaluate specificity. If there are significant genetic mutations in the BBk sample, the primers will either not anneal at all (resulting in no PCR product) or anneal but give a product that is of a different size (if the mutations are beyond the primer domain). 2. Gel set-up a. A 0.7% agarose gel was made. 10 uL of the aforementioned cocktails were loaded into each well, with the well key included in the results picture. Voltage was set at 90V and left to run for 50 minutes. A GenRuler 1 kb Plus DNA Ladder was run in parallel to provide size calibrations for our results. Results: (insert May 28 gel picture here)
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