Team:Warsaw/Calendar-Main/14 July 2009
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Ffijalkowski (Talk | contribs) (New page: {{WarNotebook}} <!-- do not edit above me! --> <!-- TU EDYTUJE FRANEK, NIE RUSZ! --> ===<div style="text-align: center;">Creating devices to test promoters in <em>E. coli</em> strains (...) |
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* Positive selection will be made according to fluorescence under UV light | * Positive selection will be made according to fluorescence under UV light | ||
<!-- TU PISZ CO CHCESZ! --> | <!-- TU PISZ CO CHCESZ! --> | ||
+ | ===Cloning p53 coding sequence=== | ||
+ | |||
+ | '''Marcin''' | ||
+ | |||
+ | |||
+ | Task 1: | ||
+ | *Transformation of chemocompetent strain of E. coli by plasmid containing p53 sequence | ||
+ | |||
+ | Procedure: | ||
+ | |||
+ | *The protocol of transformation has not been changed, except of giving 10 µl of ligated plasmid solution to the bacteria. If you want to see detailed procedure go [https://2009.igem.org/Team:Warsaw/Calendar-Main/7_July_2009 here] | ||
+ | *Bacteria was plated on the medium containing ampicillin, X-Gal and IPTG | ||
+ | |||
+ | |||
+ | Task 2: Restriction digest of pKS vector for future aplications: | ||
+ | Methods: | ||
+ | * Reaction mixture composition: 2 μl pKS plasmid, 1 μl XbaI (Fermentas), 1 μl SmaI (Fermentas) 2 μl Buffer Tango (Fermentas), 14 μl MQ water | ||
+ | |||
+ | Program: | ||
+ | |||
+ | digest: | ||
+ | <pre> | ||
+ | 1. 37°C - 8 hours | ||
+ | 2. 65°C - 15 minutes | ||
+ | </pre> | ||
+ | |||
+ | ===Assembly of endosomal detection operon=== | ||
+ | |||
+ | '''Marcin''' | ||
+ | |||
+ | Task 1: | ||
+ | *Breed bacteria to isolate plasmid containing biobricks essential to build the operon: | ||
+ | [http://partsregistry.org/Part:BBa_B0032 <span style="color: black;">BBa_B0032</span>] | ||
+ | |||
+ | [http://partsregistry.org/Part:BBa_C0040 <span style="color: black;">BBa_C0040</span>] | ||
+ | |||
+ | [http://partsregistry.org/Part:BBa_C0051 <span style="color: black;">BBa_C0051</span>] | ||
+ | |||
+ | [http://partsregistry.org/Part:BBa_E0021 <span style="color: black;">BBa_E0021</span>] | ||
+ | |||
+ | Methods: | ||
+ | # Prepare LB medium with ampicillin | ||
+ | # Add 3.5 ml of the medium to the probes | ||
+ | # Add one bacterial colony to each probe | ||
+ | # Breed the bacteria about 10 hours | ||
Revision as of 02:42, 15 July 2009
Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Tasks:
- Transformation of competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- Chemocompetent E. coli cells from Top10 strain were transformed according to our standard procedure with either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
- Tranformants with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] were plated on 18 plates with LB, agar-agar and IPTG
- Tranformants with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] were plated on 18 plates with LB, agar-agar and 0.2 % arabinose.
Results:
- Positive selection will be made according to fluorescence under UV light
Cloning p53 coding sequence
Marcin
Task 1:
- Transformation of chemocompetent strain of E. coli by plasmid containing p53 sequence
Procedure:
- The protocol of transformation has not been changed, except of giving 10 µl of ligated plasmid solution to the bacteria. If you want to see detailed procedure go here
- Bacteria was plated on the medium containing ampicillin, X-Gal and IPTG
Task 2: Restriction digest of pKS vector for future aplications:
Methods:
- Reaction mixture composition: 2 μl pKS plasmid, 1 μl XbaI (Fermentas), 1 μl SmaI (Fermentas) 2 μl Buffer Tango (Fermentas), 14 μl MQ water
Program:
digest:
1. 37°C - 8 hours 2. 65°C - 15 minutes
Assembly of endosomal detection operon
Marcin
Task 1:
- Breed bacteria to isolate plasmid containing biobricks essential to build the operon:
[http://partsregistry.org/Part:BBa_B0032 BBa_B0032]
[http://partsregistry.org/Part:BBa_C0040 BBa_C0040]
[http://partsregistry.org/Part:BBa_C0051 BBa_C0051]
[http://partsregistry.org/Part:BBa_E0021 BBa_E0021]
Methods:
- Prepare LB medium with ampicillin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- Breed the bacteria about 10 hours
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