Team:Warsaw/Calendar-Main/27 July 2009
From 2009.igem.org
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Reamplification of the mgtc promoter
Methods:
- The PCR mix was prepared as follows: 5μl buffer B (EURx), 2μl 5mM dNTPs (EURx), 5μl forward starter, 5μl reverse starter, 2μl OptiTaq polymerase (EURx), the solution was topped up with H2O to the final volume of 50μl.
- PCR programme:
4min 95°C
(30s 95°C, 35s 58°C, 40s 72°C)x28
10min 72°C
~ 4°C
- The results were visualised with gel electrophoresis on 1% agarose gel.
Results:
- Gel Electrophoresis:
From left:
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- 5μl of PCR mix
Conclusions:
- [Borat]Great success![/Borat]
Assembly of endosomal detection operon
Marcin
Task 1:
- Prepare the bacterial cultures for isolation of plasmids containing ligated biobricks
Preparation of bacterial cultures
Construction of K177012 operon1_part2
Ania
Tasks:
- extract plasmid, digest with EcoRI and PstI, gel electrophoresis
- set up liquid culture from cells transformed with repeated ligation, extract plasmid, digest with EcoRI and PstI, gel electrophoresis.
Construction of RBS.3-llo
Ania
Tasks:
- Digest vector with RBS.3 with SpeI and PstI
- Digest llo(wild type) and llo mutated with with XbaI and PstI
Results:
Comments:
The RBS.3 and wild type listeriolysin are correctly digested, the mutated listeriolysin is not digested, sequencing confirmed that we did not have the correct vesrion of mutated listeriolysin.
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