Team:Newcastle/11 August 2010

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(Difference between revisions)
(→Gibson cloning of the rocF BioBrick)
(→Transformation of E. coli DH5α cells with ligated rocF fragments)
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=Transformation of ''E. coli'' DH5α cells with ligated ''rocF'' fragments=
 
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==Aim==
 
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The aim of this experiment is to transform ''E. coli'' DH5α cells with the ligated fragments (by Gibson menthod) of ''rocF'' BioBrick so as to create multiple copies of the ligated fragments.
 
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==Materials and Protocol==
 
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Please refer to: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']].
 
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After transformation, 15 μl, 50 μl and 100 μl of transformed ''E. coli'' were plated onto 1.5% agar plate containing chloramphenicol as a selection marker.
 
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==Result==
 
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We would be putting the plates at 37°C for 48 hours and on 13th August, 2010, we would be searching for the colonies present on the plates.
 
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==Discussion==
 
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If the Gibson reaction has worked perfectly and if the transformation went successfully, then we would be having colonies on the agar plates.
 
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==Conclusion==
 
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The procedure went successfully and the result will be out by the end of 13th August, 2010.
 
=Subtilin Immunity BioBrick=
=Subtilin Immunity BioBrick=

Revision as of 00:04, 26 October 2010

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Contents

Subtilin Immunity BioBrick

Aims

Our aims for today are to run the gel electrophoresis to check whether we have the correct fragment sizes on the four parts that are we amplified yesterday. If the PCR worked, we will then perform gel extraction and then perform another gel electrophoresis for the extracted gel in order to obtain our BioBrick parts.

Newcastle Loaded Gel for Subtilin Immunity BioBrick.JPG

Materials and protocol

Please refer to the gel electrophoresis and the gel extraction protocols.

Results

Newcastle Subtilin Immunity Gel 1b.jpg

Figure 1: Gel electrophoresis of the PCR products of the parts required for the subtilin immunity BioBrick.

  • Lane 1: 1 kb DNA ladder
  • Lane 2: Plasmid Vector (pSB1C3)
  • Lane 3: Promoter and RBS (pVeg-SpoVG)
  • Lane 4: spaIFEG Gene Cluster
  • Lane 5: Double terminator
  • Lane 6: 100 bp DNA ladder

Discussion

Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5) showed up. spaIFEG PCR tube (lane 4) did not show up. We think that this occurrence was due to the Tm error (it should be 63°C as opposed to 46°C). Therefore, another PCR and gel electrophoresis were performed. Please see tomorrow's lab book page for this.

Go back to our main Lab book page

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