Team:Newcastle/26 July 2010

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Contents

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: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 E. coli DH5α with the above two plasmids, with ..., 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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