Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil

Tasks:

• Ligation to the plasmid
• Bacteria transformation

Methods:

• The ligation mix was prepared as follows:

  20μl purified plasmid backbone
  20μl mgtC promoter
  5μl LIgase Buffer (Fermentas)
  1μl T4 Ligase (Fermentas)
  4μl H2O

• The ligation was carried out in 18°C for 4h.
• A fresh batch of chemocompetent bacteria was transformed according to the protocol.

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Ligation to the plasmid
• Bacteria transformation

Methods:

• The ligation mix was prepared as follows:

  20μl purified plasmid backbone
  10μl Cro-Box
  4μl LIgase Buffer (Fermentas)
  5μl 30% PEG
  1μl T4 Ligase (Fermentas)

• The ligation was carried out in 18°C for 4h.
• A fresh batch of chemocompetent bacteria was transformed according to the protocol.

Assembly of fusion proteins

Marcin

Task 1: Preparation of bacterial cultures to isolate mitochondrial peptide on pSB1A3 plasmid

One important remark:

Data from sequencing reactions clearly show that previously isolated plasmid which should contain: mitochondrial peptide CDS (BBa_K177028 - blunt ligation with pKSII) and BBa_K177037 are other commercial plasmids. Something is wrong - some plasmids samples have been contaminated.