Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil

Tasks:

• Amplification of the mgtc promoter using new primers
• Isolation of the pSB1A3 plasmid

Methods:

• The PCR mix was prepared as follows:

  5μl buffer B (EURx)
  5μl 10mM dNTPs
  5μl forward starter
  5μl reverse starter
  2μl OptiTaq polymerase (EURx)
   2μl Salmonella matrix
   26 μl H2O

• PCR programme:

   4min 95°C 
   (30s 95°C, 35s 56°C, 40s 72°C)x28 
   10min 72°C 
   ~ 4°C
  

• The PCR results were visualised with gel electrophoresis on 1% agarose gel.
• The plasmid isolation was carried out from 3μl of culture cultivated for 6h in 37°C. The Plasmid Mini kit (A&A Biotechnology) was used.

Results:

• Nothing

Conclusions:

• The temperature was chosen... poorly.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Creation of the cro-box double stranded DNA

Methods:

• The mixture of equal volumes (10μl) of both ssDNA was heated to 94°C for 15min. and left to cool.
  

Assembly of endosomal detection operon

Marcin

Task 1:

• Digest of following constructs:
• BBa_C0051 with BBa_B0032 on pSB1A3 plasmid
• BBa_E0022 with BBa_B0032 on pSB1A3 plasmid

Methods:

• Reaction mixture composition:

 
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas) in the case of C0051
0.7 μl SpeI (Fermentas) in the case of E0022
2 μl Buffer Tango (Fermentas)
15 μl MQ water

• Digest was performed about seven hours
• After digestion samples were frozen

Making of the plac-RBS-llo-intA part

Jarek

Tasks:

• Separation of digested DNA fragments in 0,8% agarose.

Results:

• The intA gene that was used might not be the intA gene after all.

Cloning switch 1 regulatory parts [ K177012-PcI.RBS.LacI, K177033-PcI.RBS.LacI.PcI.RBS.RFP.terminator, K177011-PLacI.RBS.cI.terminator, K177038-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator ] into two compatible low copy number plasmids of different antibiotic resistance

Ania

Tasks: