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:

1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. 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

• Prepare LB medium with kanamycin
• Add 3.5 ml of the medium to the probes
• Add one bacterial colony to each probe
• Breed the bacteria about 7 hours

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-listeriolysin

Ania

Tasks:

• Digest vector with RBS.3 with SpeI and PstI
• Digest llo(wild type) and llo mutated with with XbaI and PstI
• Extract the correct fragments from the gel
• Set up a ligation of RBS.3 and wild type llo (since the mutated version turned out not to be correct one)

Results:

Comments: