Cloning of hly gene into pKSII+ vector

Kama

Tasks:

• Amplification of hly

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, ale KNGiE też powinna działać) 
  1μl template DNA from Listeria

  Solution was topped up with H2O to 25μl.

  1 repeat of every sample was made (2 different programs).

  Additionally for each sample was made version with 10∗ dilution of template.

• PCR programs:

hly

4min 95°C 
 (30s 95°C, 35s 42°C, 1min20s 72°C)x3 
 (30s 95°C, 35s 47°C, 1min20s 72°C)x28 
 10min 72°C 
 ~ 7°C

random program

• Electrophoretic separation on 1% agarose gel

Results: