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Team:Warsaw/Calendar-Main/4 August 2009
From 2009.igem.org
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Cloning of the mgtc promoter into the pSB1A3 plasmid
Monika
Tasks
- Plasmid assembly
Methods
- The plasmid digest mix contained: 4μl purified plasmid, 2μl Tango buffer (Fermentas), 0.5μl SpeI enzyme, 0.5μl XbaI enzyme, the solution was topped up with H2O to the final volume of 20μl.
- The mgtc promoter digest mix contained: 12μl purified promoter, 3μl Tango buffer (Fermentas), 0.5μl SpeI enzyme, 0.5μl XbaI enzyme, the solution was topped up with H2O to the final volume of 30μl.
- The digest was carried out in 37°C for 3h, but 1μl CIAP enzyme was added 1h before end to mgtc promoter digest mix 1h before the end of incubation. Then enzymes were inactivated in 80°C for 20min.
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmid containing BBa_B0032 and BBa_C0040 from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Methods:
Task 2:
- Digest of isolate plasmids with ligated biobricks to verify the success of ligation
Methods:
- Digest of isolate plasmids using XbaI and PstI
- Reaction mixture composition: 0.5 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl PstI (Fermentas), 2 μl Buffer Tango (Fermentas), 16.5 μl MQ water
- The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analyse restriction pattern of the plasmids
Results:
verification of the restriction patterns
Comment:
All isolated plasmids do not have insert. The cloning must be performed another time
Task 3:
- Another Cloning of this biobrick
Methods:
- Ligation mixture composition: 11 μl both digested fragments, 2.5 μl ligation buffer (Fermentas), 1 μl ligase T4 (Fermentas)
- Duration of ligation was about 16 hours
Cloning of p53 coding sequence
Marcin
Task 1:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Detailed protocol of transformation is described here. The only modification is usage of total volume of ligation mixture prepared 03.08.09
- petri dish will be held in 37°C for 16 hours
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