Team:Newcastle/11 August 2010

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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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=Gel electrophoresis for the subtilin immunity fragments=
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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.
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=Subtilin Immunity BioBrick=
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==Aims==
==Aims==
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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 [[Team:Newcastle/10 August 2010#Subtilin_Immunity_BioBrick| 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.
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The aim of the experiment is to check whether we have the correct fragment sizes on the four subtilin fragments that are we have amplified [[Team:Newcastle/10 August 2010#Subtilin_Immunity_BioBrick| yesterday]].
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[[Image:Newcastle_Loaded_Gel_for_Subtilin_Immunity_BioBrick.JPG|right|200px|thumb]]
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==Materials and protocol==
==Materials and protocol==
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Please refer to the [[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]] and the [[Team:Newcastle/Gel_extraction| gel extraction]] protocols.
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Please refer to the [[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]] protocol.
==Results==
==Results==
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==Discussion==
==Discussion==
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Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5) showed up.
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The correct band size were observed for for the Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5). However no band was observed  for the ''spaIFEG''(lane 4).  
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''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 [[Team:Newcastle/12_August_2010| tomorrow's]] lab book page for this.
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==Conclusion==
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No band was observed for the ''spaIFEG''. This could be due to the wrong melting temperature.
'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 00:11, 26 October 2010

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Contents

Gel electrophoresis for the subtilin immunity fragments

Aims

The aim of the experiment is to check whether we have the correct fragment sizes on the four subtilin fragments that are we have amplified yesterday.

Materials and protocol

Please refer to the gel electrophoresis protocol.

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

The correct band size were observed for for the Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5). However no band was observed for the spaIFEG(lane 4).

Conclusion

No band was observed for the spaIFEG. This could be due to the wrong melting temperature.

Go back to our main Lab book page

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