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 pSB1A3 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

• pSB1A3 plasmid was previously prepared as described here

Assembly of endosomal detection operon

Marcin

Task 1:

• Gel-out of BBa_C0040 with BBa_B0032 on pSB1A3 plasmid

Methods:

• sample was inactivated via heating in 80 °C for 20 minutes
• Gel-out was performed using the EurX gel-out kit according to the manual

Results:

Task 2:

• Ligate BBa_C0040 with BBa_B0032 to pSB1A3 plasmid with BBa_R0080

Methods:

• Reaction mixture composition:

10 μl insert
7 μl vector
2.3 μl Buffer Tango (Fermentas)
3 μl dNTPs mixture (EurX)
1 μl T4 ligase (Fermentas)

• Ligation was performed for about 12 hours and it was subsequently thermally inactivated

Task 3:

• Transformation of chemocompetent E. coli strain DH5&alpha

Constructs to transform:

• p53 CDS + BBa_B0032
• cro CDS + pSB1A3 plasmid

Methods:

• Detailed protocol of transformation is described here.