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Team:Newcastle/26 July 2010
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Preparation for cloning of the rocF BioBrick
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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:pSB1AK3 pSB1AK3] with [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 separate .. of E. coli DH5α with the above two plasmids, with [http://partsregistry.org/Part:BBa_K143062 BBa_K143062] (LacI BioBrick sent to us by Imperial, we will using this to help us characterise many of our BioBricks, including rocF) and with a control which we had prepared from our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with rfp insert.
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.
   
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