Protocol

Abbreviation

20

Name

20

Laboratory name

University of Calgary

Type

cloning

Description

To be used as a ligation independent cloning vector, 15 bp addition to 5¿end of primers

Fwd Primer: 5¿-GGA GAT ATA CAT ATG-----3¿

Rev Primer: 5¿-GTG GTG GTG CTC GAG-----3¿

Remove start and stop codons from the sequence to be inserted.

Contains 2kb sacB gene between the BseRI cloning sites.

pET21a-LIC used as a backbone. N-term his-tag was removed (NdeI digestion) and multiple cloning site+stop codon before the C-term His-tag was removed too (XhoI digestion). SacB was taken from pET28GST-LIC and the amino acids (LE) will be between the protein and the His-tag. Both digestion sites (NdeI and XhoI) were reconstructed.