Team:Newcastle/10 September 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Sending off ''yneA'' BioBrick=
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=Characterization of ''yneA''=
==Aim==
==Aim==
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The aim of this experiment is to extract ligated ''yneA'' fragment in the plasmid pSB1C3 from ''E. coli'' cells and purify them and freeze dry them to send them off to the iGEM headquarters.
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The aim of this experiment is to prepare slides for the ''Bacillus subtilis'' 168 cells which have ''yneA'' on the plasmid (pMutin4 and pMap65) and under ''lacI'' regulation and are induced by IPTG at various concentrations.
==Materials and Protocol==
==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Qiagen Minipreps|Qiagen Minipreps]] for plasmid extraction from ''E. coli'' cells.
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Please refer to: [[Team:Newcastle/IPTG INduction|Slide preparation for IPTG induced cells]] for materials and protocol.
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For this experiment we used the following cell colonies:
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#''Bacillus subtilis'' 168
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#''Bacillus subtilis'' 168 with pMutin4 having ''yneA'' insert
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#''Bacillus subtilis'' 168 tith pMap65 having ''yneA'' insert
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Please refer to: [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] to check the purity of the extracted and purified plasmid.
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Today we see the cells on a flourescent microscope.
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==Result==
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{|border=1
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|-
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!'''Tube'''
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!'''Nanodrop Value (in ng/ml)'''
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|-
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|1
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|76.5
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|-
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|2
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|61.5
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|-
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|3
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|84.6
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|-
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|4
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|19.5
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|-
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|5
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|89.9
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|-
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|6
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|87.6
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|-
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|7
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|6.3
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|-
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|8
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|69.5
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|-
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|9
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|81.3
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|-
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|10
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|74.4
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|-
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|11
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|68.5
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|-
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|12
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|70.2
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|-
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|13
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|83.5
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|-
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|14
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|77.6
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|-
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|15
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|73.3
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|-
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|16
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|63.0
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|}
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Nanodrop results for pSB1C3 + ''yne''A:
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#158.7
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#261.2
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#262.9
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#204.7
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#262.9
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#202.1
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#174.8
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#226.6
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#229.1
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#164.5
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#177.8
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#225.1
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#281.5
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#249.3
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#192.2
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#239.3
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#195.1
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=Arabinose-inducible promoter characterisation=
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...
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==Miniprep of BBa_I13401 (GFP coding sequence + terminator)==
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..from last night's overnight cultures from the 8th of september transformation.
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==Double digests==
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.. double digests for standard BioBrick assembly.. cut the arabinose-inducible promoter BioBrick with EcoRI and SpeI, cut gfp+terminator BioBrick with EcoRI and XbaI, for later ligation..
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==Gel extraction of digests==
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==Ligation==
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==Result and Conclusion==
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Please refer to: [[Team:Newcastle/Filamentous_Cells#Characterisation|Characterisation]] for the result.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 03:14, 26 October 2010

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Contents

Characterization of yneA

Aim

The aim of this experiment is to prepare slides for the Bacillus subtilis 168 cells which have yneA on the plasmid (pMutin4 and pMap65) and under lacI regulation and are induced by IPTG at various concentrations.

Materials and Protocol

Please refer to: Slide preparation for IPTG induced cells for materials and protocol. For this experiment we used the following cell colonies:

  1. Bacillus subtilis 168
  2. Bacillus subtilis 168 with pMutin4 having yneA insert
  3. Bacillus subtilis 168 tith pMap65 having yneA insert

Today we see the cells on a flourescent microscope.

Result and Conclusion

Please refer to: Characterisation for the result.

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