Team:Newcastle/26 July 2010

Preparation for cloning of the rocF BioBrick

Aim

In preparation for Gibson cloning of the rocF BioBrick we started work on mini preps of plasmid DNA, and on B. subtilis 168 chromosomal DNA extraction.

Re-hydration of registry parts

Re-hydration of dried parts registry DNA

We re-hydrated:

1. [http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and
2. [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.

Transformation of E. coli

We transformed and plated separate tubes of E. coli DH5α with:

1. The above two re-hydrated plasmids
2. [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial which we will using this to help us characterise many of our BioBricks, including rocF.
3. A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with rfp insert.
4. A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).

Please see transformation protocol.

Overnight cultures of B. subtilis 168 for chromosomal DNA extraction

The rocF coding sequence is to be taken from the B. subtilis 168 genome by PCR. Before we can do this we need to extract 168 chromosomal DNA.

Today we plated up overnight cultures of B. subtilis 168 so that we can do chromosome extraction tomorrow.

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.