Team:Newcastle/26 July 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=='''Preparation for cloning of the ''rocF'' BioBrick'''==
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='''Preparation for cloning of the ''rocF'' BioBrick'''=
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===Aim===
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The aim of our experiment today is to tranform ''E. coli'' DH5α cells with pSB1C3, pSB1AK3 (plamid consisting ''lacI'' and  plasmid consisting double terminator.
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===Material Required===
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==Aim==
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====For the Transformation Experiment====
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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.
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# Sterile Distill Water
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# Gilson Pipette
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# Biobrick Parts Distribution Kit
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==Re-hydration of registry parts==
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===Re-hydration of registry parts===
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[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]
[[Image:Newcastlehydration.jpg|200px|thumb|right|Re-hydration of dried parts registry DNA]]
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We re-hydrated:  
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We re-hydrated using sterile distill water:  
#[http://partsregistry.org/Part:pSB1C3 pSB1C3] (the plasmid we will be submitting our BioBricks to the registry in) and
#[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.
#[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.
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===Transformation of ''E. coli''===
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==Transformation of ''E. coli''==
We transformed and plated separate tubes of ''E. coli'' DH5α with:
We transformed and plated separate tubes of ''E. coli'' DH5α with:
# The above two re-hydrated plasmids
# The above two re-hydrated plasmids
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# [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''.
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# [http://partsregistry.org/Part:BBa_K143062 BBa_K143062], a LacI BioBrick sent to us by Imperial College, London, UK which we will use to help characterise many of our BioBricks, including ''rocF''.
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.
# A positive control which we had already prepared during our training week - [http://partsregistry.org/Part:pSB1AT3 pSB1AT3] with ''rfp'' insert.
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).
# A negative control (no vector), to verify the antibiotic plates are working (no growth should be observed on this plate).
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Please see transformation protocol.
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Please refer to the transformation protocol for ''E. coli'' DH5α here: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].
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===Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction===
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==Overnight cultures of ''B. subtilis'' 168 for chromosomal DNA extraction==
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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.
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The ''rocF'' coding sequence is to be amplified 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.
Today we plated up overnight cultures of ''B. subtilis'' 168 so that we can do chromosome extraction tomorrow.
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==Colony PCR of Genomic DNA==
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='''PCR of Genomic DNA'''=
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===Aim:===
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==Aim:==
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To determine whether the genes have been inserted into the plasmid of ''B. subtilis 168''.
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To determine whether the genomic DNA has been extracted from ''B. subtilis'' strains 168 and 3610.
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===Materials:===
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==Materials:==
* Pipette
* Pipette
* Microfuge
* Microfuge
* Microtubes
* Microtubes
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* Distilled H2O
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* Distilled H<sub>2</sub>O
* Nucleotide DNTP
* Nucleotide DNTP
* 5x GoTaq buffer
* 5x GoTaq buffer
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* Template DNA (B. Subtilis ATCC 6633, 1:1 and 1:2)
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* Template DNA
* Forward and reverse primers
* Forward and reverse primers
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===Protocol:===
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==Protocol:==
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* For the full protocol, please refer to [[TeamNewcastleColony_PCR|Colony PCR]] in Protocol List.
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* For the full protocol, please refer to [[Team:Newcastle/PCR|PCR]].
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====Conditions in ThermoCycler:====
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===Conditions in ThermoCycler:===
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* Melting temperature, Tm used for Anneal step is 59°C.
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* Melting temperature, Tm used for anneal step is 59°C.
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===Results:===
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==Results:==
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Gel electrophoresis will be run tomorrow to determine the results.
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Gel electrophoresis will be undertaken tomorrow to determine the results.
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===Conclusion:===
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==Conclusion:==
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Please refer to Lab book dated 27.7.2010.
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Please refer to Lab book dated [[Team:Newcastle/27_July_2010|27th July 2010]].
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 21:12, 27 October 2010

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Contents

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 using sterile distill water:

  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 College, London, UK which we will use to help 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 refer to the transformation protocol for E. coli DH5α here: Transformation of E. coli.

Overnight cultures of B. subtilis 168 for chromosomal DNA extraction

The rocF coding sequence is to be amplified 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.

PCR of Genomic DNA

Aim:

To determine whether the genomic DNA has been extracted from B. subtilis strains 168 and 3610.

Materials:

  • Pipette
  • Microfuge
  • Microtubes
  • Distilled H2O
  • Nucleotide DNTP
  • 5x GoTaq buffer
  • Template DNA
  • Forward and reverse primers

Protocol:

  • For the full protocol, please refer to PCR.

Conditions in ThermoCycler:

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

Results:

Gel electrophoresis will be undertaken tomorrow to determine the results.

Conclusion:

Please refer to Lab book dated 27th July 2010.

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