PCR inv

Kama

Tasks:

• Amplification of inv

Methods:

• PCR mixture's composition:

  2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul 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, 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:

Insertion of the hly gene into the pKSII+ plasmid

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 fallows: 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 fallows: 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: