PCR inv

Kama

Tasks:

• Amplification of inv

Methods:

• PCR mixture's composition:

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

  Solution was topped up with H2O to 25μl.

• PCR program:

inv

4min 95°C 
 (30s 95°C, 35s 58°C, 4min15s 72°C)x3 
 (30s 95°C, 35s 63°C, 4min15s 72°C)x28 
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Cloning of hly gene into pKSII+ vector

Kamil

Tasks:

• Purification of the PCR product
• Plasmid assembly

Methods:

• The purification was carried out using Montage PCR Centrifugal Filter Device P36461 (Milipore) and according to the protocol supplied.

• Plasmid digest mix was prepared as follows:

  2ul Tango buffer (Fermentas) 
  1ul pKSII plasmid 
  1ul XbaI enzyme 
  1ul SmaI enzyme

  The solution was topped up with H2O to the final volume of 20 ul.
• hly gene digest mix was prepared as follows:

  2ul Tango buffer (Fermentas) 
  2ul purified gene 
  1ul XbaI enzyme

  the solution was topped up with H2O to the final volume of 20 ul.
• The digest was kept for 3h at 37°C, and then the enzyme was inactivated for 15min. at 80°C.

• The ligation mix was prepared as follows:

  1ul plasmid 
  1ul gene 
  1ul ligation buffer G (Fermentas) 
  1ul T4 DNA ligase (Fermentas)

  The solution was topped up with H2O to the final volume of 10 ul. The ligation was carried out in 18°C overnight (~12h) and the inactivated for 10min. at 65°C.

• Electrophoretic separation on 1% agarose gel

Results: