Amplyfing of Pho sequence

Justyna

Methods

• PCR reaction mix:

per 50μl:

5.0 μl - 10 x buffer (20mM MgSO4)
3.5 μl - dNTP mix (5mM)
2.5 μl - primer PhoF 
2.5 μl - primer PhoR2 
3.75μl - DMSO
2.0 μl - template DNA
28.25μl - mQ water
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)



• buffer, dNTP mix and polymerase from EURx.
• DNA template - Salmonella genomic DNA




• PCR reaction has been done in two extension temperatures - 68°C and 72°C

Thermal cycling conditions for PCR:




94°C, 1 min 0s

94°C, 0 min 15s
55°C, 0 min 30s
68°C / 72°C (either), 1 min 0s
cycles 1-25

68°C / 72°C (either), 7 min 0s
4°C, indefinite




Results:

• Effective reaction.

Cloning Pho into pSB plasmid

Justyna

Task 1:

• Gel-out Pho PCR product

Methods:

• Pho PCR products were isolated from agarose gel using A&A Gel-Out kit.



• The arrow points the right and best isolated product

• pSB plasmid was previously prepared as described here