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Team:Warsaw/Calendar-Main/21 September 2009
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(New page: {{WarNotebook}} <!-- do not edit above me! --> <h3>Assembly of endosome detection operon</h3> <h4>Marcin</h4> Task 1: Isolation of [http://partsregistry.org/Part:pSB1A3<span style="color:...) |
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Methods: | Methods: | ||
* Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here] | * Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here] | ||
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Task 2: Restriction digest of previously isolated plasmids: | Task 2: Restriction digest of previously isolated plasmids: | ||
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Some sequences 1500 bp length are visible on the gel but I need to determine whether it is expected product or contamination (bacteria containing plasmid with listeriolysin were also presented on the same plate) | Some sequences 1500 bp length are visible on the gel but I need to determine whether it is expected product or contamination (bacteria containing plasmid with listeriolysin were also presented on the same plate) | ||
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Task 3: PCR reaction to determine the identity of isolated (Task 1) DNA | Task 3: PCR reaction to determine the identity of isolated (Task 1) DNA | ||
| - | '''Commment''': I decided to perform PCR reaction using primers for listeriolysin. If the plasmids contain [http://partsregistry.org/Part:BBa_K177044<span style="color: black">BBa_K177044</span>] there will be no PCR product. | + | '''Commment''': |
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| + | I decided to perform PCR reaction using primers for listeriolysin. If the plasmids contain [http://partsregistry.org/Part:BBa_K177044<span style="color: black">BBa_K177044</span>] there will be no PCR product. | ||
Methods: | Methods: | ||
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All isolated plasmid contain listeriolysin. It is necessary to carry out ligation one more time | All isolated plasmid contain listeriolysin. It is necessary to carry out ligation one more time | ||
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Task 4: | Task 4: | ||
Revision as of 12:46, 24 September 2009
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Assembly of endosome detection operon
Marcin
Task 1: Isolation of [http://partsregistry.org/Part:pSB1A3pSB1A3] plasmid containing [http://partsregistry.org/Part:BBa_K177044BBa_K177044]
Methods:
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here]
Task 2: Restriction digest of previously isolated plasmids:
Methods:
- Reaction mixture composition:
3 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 13 μl MQ water
- Reactions were carried out 2 hour
Results:
Some sequences 1500 bp length are visible on the gel but I need to determine whether it is expected product or contamination (bacteria containing plasmid with listeriolysin were also presented on the same plate)
Task 3: PCR reaction to determine the identity of isolated (Task 1) DNA
Commment:
I decided to perform PCR reaction using primers for listeriolysin. If the plasmids contain [http://partsregistry.org/Part:BBa_K177044BBa_K177044] there will be no PCR product.
Methods:
- Reaction mixture composition:
0.1 μl purified plasmid DNA product 1.4 μl Opti Taq Polymerase (EURx) 1 μl dNTPs (EURx) 2 μl Opti Taq Buffer (Fermentas) 15.5 μl MQ water
Results:
All isolated plasmid contain listeriolysin. It is necessary to carry out ligation one more time
Task 4:
- Transformation of chemocompetent E. coli TOP10 strain
Construct to transform:
- [http://partsregistry.org/Part:BBa_K177044BBa_K177044]
Methods:
- Ligation mixture was thermally inactivated in 80 °C for 20 minutes
- Detailed protocol of transformation is described here
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