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Team:Warsaw/Calendar-Main/20 August 2009
From 2009.igem.org
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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)
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:
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:
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:
Result of the digestion
Task 2:
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)
Task 3:
- Transformation of chemocompetent E. coli strain DH5&alpha
Constructs to transform:
Methods:
- Detailed protocol of transformation is described here.
Making of the plac-RBS-llo part
Jarek
Tasks:
- Separation of restriction fragments in 0,8% agarose gel
- Digestion of 5 samples of plasmid DNA acquired on 19 August with PvuII and PstI restriction endonucleases
Ania
Tasks:
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