Preparation of constructs with OmpA protein fusions

Piotr

1. Transformation of E. coli TOP10 strain with ligation from previous day.
2. Transformants plating on LB + kanamycin.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

1. Gel electrophoresis of PCR products
2. Gel-out of proper bands
3. PCL reaction to create omega-A fusion:
   
   

   Product	Templates	Primers	Product length
   Omega-A fusion	Omega-linker + linker-A	OmegaL+SacI + AP+NotI	1440 bp

   
   

   PCL program for omega-A fusion
   Temperature	Time
   94°C	4:00
   94°C	0:30	28 cycles
   48°C-58°C	0:45
   68°C	2:00
   68°C	10:00
   4°C	infinite

   
   
4. Gel electrophoresis of PCL products
5. We have obtained no PCL product (weird?)

Preparation of construct pKS with A protein
Michał L., Marcin:

1. Inactivation of digestion enzymes and CIAP.
2. Ligation of digested PCR product and pKS 2h at room temperature.
3. Transformation of E. coli TOP10 strain with 7 µl of ligation mix.
4. Transformants plating on LB + ampicillin + X-gal + IPTG.