Team:Warsaw/Calendar-Main/11 September 2009

Assembly of fusion proteins Marcin Task 1: Prepare the ligation of PCR-amplified signal peptide with pKSII cloning vector Methods: 11 &mu;l amplified signal peptide CDS (previously phosphorylated via PNK) 7 &mu;l vector 1 &mu;l T4 ligase (Fermentas) 2.5 &mu;l dNTPs mixture (EurX, concentration 5 mM) 2.5 &mu;l Buffer Tango (Fermentas) 2 &mu;l PEG 4500 (Fermentas)
 * Reaction mixture composition:
 * Ligation was carried out 12 hours in 16 &deg;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 11 &mu;l insert 8 &mu;l vector 1 &mu;l T4 ligase (Fermentas) 2.5 &mu;l Buffer Tango (Fermentas) 2.5 &mu;l dNTPs mixture (EurX, concentration 5 mM)
 * Reaction mixture composition:
 * Ligation was carried out 12 hours in 16 &deg;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