Assembly of endosomal detection operon

Marcin

Task:1

• Transformation of chemocompetent E. coli TOP10 strain

Constructs to transform:

• Bax CDS + BBa_B0032
• phoP/Q CDS + pSB1A3 plasmid

Methods:

• Detailed protocol of transformation is described here

Task 2:

• Digest isolated plasmid with Bax CDS to verify the correctness of the ligation:

Methods:

• Reaction mixture composition:

2 μl purified plasmid DNA product
1 μl RsaI (NEB)
2 μl Buffer Tango (Fermentas)
15 μl MQ water

• Digest was performed 3 hours

Results:

Comments:

• The enzym must have been deactivated due to presence of two forms of the plasmid. If circular plasmid is cut there are only one linear form in the sample. Althought in each case there was two forms - circular and supercoiled.