MutD₅ testing

Piotr

Inoculation MutD₅ from previous day to the same medium (liquid LB + 0.25mM IPTG + kanamycin + 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 BioBricks

Piotr

1. E. coli TOP10 transformation with overnight ligation of RFP(??????) and deltaA DNA fragments.
2. Plating on LB + ampicillin.