"
Team:Warsaw/Calendar-Main/6 May 2009
From 2009.igem.org
(Difference between revisions)
(New page: {{WarNotebook}} <!-- do not edit above me! --> <emptyday> <!-- do not remove this! --> {{WarNotebookEnd}}) |
|||
| Line 4: | Line 4: | ||
| + | |||
| + | |||
| + | ===Cloning of the listeriolysin gene=== | ||
| + | |||
| + | '''Marcin''' | ||
| + | |||
| + | Task | ||
| + | *Ligation of the listeriolysin gene to the pKS plasmid | ||
| + | |||
| + | Methods: | ||
| + | *Evaluation of the quality of the used DNA samples: | ||
| + | Concentration and quality of used DNA was verify via electrophoresis of DNA sample with MassRuler DNA ladder (Fermentas) and GeneRuler DNA ladder (Fermentas). 1 μl of pKS plasmid and insert were diluted to 10 μl and loaded into gel | ||
| + | |||
| + | [[image:Evaluation_06_05_09.png|thumb|centre|300px|Evaluation of the insert and vector DNA samples]] | ||
| + | |||
| + | *Ligation of the pKS plasmid and listeriolysin gene: | ||
| + | Proper ligation mixture composition: 2.5 μl insert solution, 8 μl plasmid solution, 4 μl ligation buffer (Invitrogen, contain PEG4500), 1 μl T4 ligase (Fermentas), 4.5 μl MQ water | ||
| + | |||
| + | Control ligation mixture composition: the same as proper mixture except lack of insert solution | ||
| + | |||
| + | Ligation was took place in room temperature during 6 hours. Ligase was subsequently inactivated by heating to 80 °C for 15 minutes. After inactivation both mixtures were frozen. | ||
| - | |||
Revision as of 18:30, 12 July 2009
 |  |
 |
  |
 |
|
Cloning of the listeriolysin gene
Marcin
Task
- Ligation of the listeriolysin gene to the pKS plasmid
Methods:
- Evaluation of the quality of the used DNA samples:
Concentration and quality of used DNA was verify via electrophoresis of DNA sample with MassRuler DNA ladder (Fermentas) and GeneRuler DNA ladder (Fermentas). 1 μl of pKS plasmid and insert were diluted to 10 μl and loaded into gel
- Ligation of the pKS plasmid and listeriolysin gene:
Proper ligation mixture composition: 2.5 μl insert solution, 8 μl plasmid solution, 4 μl ligation buffer (Invitrogen, contain PEG4500), 1 μl T4 ligase (Fermentas), 4.5 μl MQ water
Control ligation mixture composition: the same as proper mixture except lack of insert solution
Ligation was took place in room temperature during 6 hours. Ligase was subsequently inactivated by heating to 80 °C for 15 minutes. After inactivation both mixtures were frozen.
|
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
