Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

We have finally got rid of evil phage infection and we are able to work with E. coli again. Hurray!

1. Since the phage is gone we can continue this cloning:
2. Restriction digest of PCL product and pKS vector with SacI and NotI (BamHI buffer) .
3. Ligation of omega-A fusion with pKS vector.
4. Transformation of Top10 with ligation product.

Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA

Paweł

1. Result of ligation of pET15b-OmpA-omega with protein Z DNA: 4 colonies grown.
2. Each colony cultured overnight in LB + ampicilin.

Cloning of protein A DNA to OmpA constructs

Michał K.

1. Digest of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (BamHI buffer). pACYC177 vectors were also dephosphorylated (with CIAP).
2. Gel electrophoresis and gel-out of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane) (Fig. 1.).
3. Ligation of A DNA fragment with both pACYC177 vectors.
4. Transformation of E. coli TOP10 strain with ligations.
5. Transformants plating on LB + kanamycin.