Assembly of fusion proteins

Marcin

Task 1: Prepare the ligation of PCR-amplified signal peptide with pKSII cloning vector Methods:

• Reaction mixture composition:

11 μl amplified signal peptide CDS (previously phosphorylated via PNK)
7 μl vector
1 μl T4 ligase (Fermentas)
2.5 μl dNTPs mixture (EurX, concentration 5 mM)
2.5 μl Buffer Tango (Fermentas)
2 μl PEG 4500 (Fermentas)

• Ligation was carried out 12 hours in 16 °C. After mixture was thermally inactivated

Task 2: Transformation of TOP10 chemocompetent bacteria with mitochondial signal peptide CDS

Methods:

• Ligation mixture was thermally inactivated
• Detailed protocol of transformation is described here

Assembly of endosome detection operon

Marcin

Task 1: Prepare the ligation BBa_K177035 with BBa_K177036 to obtain BBa_K177037

• Reaction mixture composition:

11 μl insert
8 μl vector
1 μl T4 ligase (Fermentas)
2.5 μl Buffer Tango (Fermentas)
2.5 μl dNTPs mixture (EurX, concentration 5 mM)

• Ligation was carried out 12 hours in 16 °C. After mixture was thermally inactivated

Task 2: Transformation of TOP10 chemocompetent bacteria with mitochondial signal peptide CDS

Methods:

• Ligation mixture was thermally inactivated
• Detailed protocol of transformation is described here