Team:Newcastle/26 July 2010

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Contents 1 Preparation for cloning of the rocF BioBrick 1.1 A 1.2 Overnight cultures of B. subtilis 168 for chromosomal DNA extraction 2 Colony PCR of Genomic DNA 2.1 Aim: 2.2 Materials: 2.3 Protocol: 2.3.1 Conditions in ThermoCycler: 2.4 Results: 2.5 Conclusion:

Preparation for cloning of the rocF BioBrick

A

• Re-hydrated [http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] (the double terminator we will be using for the rocF BioBrick) from the parts distribution.
• Transformed and plated E. coli DH5α

Overnight cultures of B. subtilis 168 for chromosomal DNA extraction

Colony PCR of Genomic DNA

Aim:

To determine whether the genes have been inserted into the plasmid of B. Subtilis 168.

Materials:

• Pipette
• Microfuge
• Microtubes
• Distilled H2O
• Nucleotide DNTP
• 5x GoTaq buffer
• Template DNA (B. Subtilis ATCC 6633, 1:1 and 1:2)
• Forward and reverse primers

Protocol:

• For the full protocol, please refer to Colony PCR in Protocol List.

Conditions in ThermoCycler:

• Melting temperature, Tm used for Anneal step is 59°C.

Results:

Gel electrophoresis will be run tomorrow to determine the results.

Conclusion:

Please refer to Lab book dated 27.7.2010.