MutD₅ testing

Emilia

1. Inoculation MutD₅ from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)

2. 6 hours culture
3. Inoculation MutD₅ from the morning to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)

Mutagenesis of protein A

Paweł

1. Results of mutagenesis: no colonies on any plate.
2. Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.

Preparation of alpha_A construct

Antoni

1. PCR on pKS plasmid containing protein A with AL+link10+homo2 and AP+NotI primers and 10% DMSO(20 cycles, elongation 40 s, annealing temperature 72°C).
2. PCR on pUC19 plasmid with AlphaL+SacI and AlphaP+link10+homo2 primers (20 cycles, elongation 45 s, annealing temperature 63°C).
   As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR.
3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker - 570 bp and linker_A - 470 bp ).
4. Measurment of concentration of both isolated products.

Preparation of ΔA (BBa_K103003)

Piotr

1. E. coli TOP10 transformation with overnight ligation of pSB1A3 and ΔA DNA fragments.
2. Plating on LB + ampicillin.