PCR inv

Kamil

Tasks:

• Amplification of inv

Methods:

• PCR mixture's composition:

  2,5ul pfu buffer (Fermentas) 
  2,5ul MgSO4 (Fermentas) 
  1,5ul primers 
  1ul dNTPs (10 mM) 
  0,5ul pfu turbo polymerase (od Antka) 
  1ul template DNA from Yersinia

  Solution was topped up with H2O to 25ul.

• PCR program:

inv

4min 95°C 
 (30s 95°C, 1min 53°C, 4min15s 72°C)x3 
 (30s 95°C, 1min 58°C, 4min15s 72°C)x28 
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Cloning of hly gene into pKSII+ vector

Marcin

Task:

• Transformation of electrocompetent E. coli strain TOP10 with pKS plasmid containing hemolysin coding sequence

Procedure:

1. Ligation mixture (9 μl) was dialise 30 minut
2. Half of the volume of the mixture was added to thawed bacteria
3. Bacteria were transfered to the cuvette and electroporated (2500V, 5 sec)
4. After the electroporation 1 ml of SOB medium was added to the cuvette
5. Mixture with bacteria was transfered to the Eppendorf probe and it was incubated for 45 minut in 37 °C
6. Mixture was divided to 2 equal portion which were plated on medium containing ampicillin, X-Gal and IPTG