Today we checked for transformants from the ''Bacillus subtilis'' transformation (with pMutin4) we performed yesterday.
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===Aims===
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The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of ''rfp'' on the pSB1C3 plasmid seperately. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] for Phusion PCR protocol.
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The details of the four PCR reactions are included in the table below:
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{|border=1
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|-
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!'''Tube'''
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!'''Part to be amplified'''
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!'''DNA fragment consisting the part i.e. template'''
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!'''Forward primer'''
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!'''Reverse Primer'''
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!'''Melting Temperature (Tm in °C) '''
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!'''Size of the fragment (in bp)'''
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!'''Extension time (in seconds)'''
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|-
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|1
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|Hyperspankoid
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|yneA
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|Prom_for
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|HpSpanPspacoid_rev
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|65
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|150
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|15
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|-
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|2
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|Pspacoid
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|kinA
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|Prom_for
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|HpSpanPspacoid_rev
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|65
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|150
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|15
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|-
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|3
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|Hyperspank
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|K143055 from Bacillus plate - BS022
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|Prom_for
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|Pspac-hy rev
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|62
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|150
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|15
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|-
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|4
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|Plasmid
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|vector pSB1C3/''rfp''
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|Vector/RFP_for
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|P2-V1
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|64
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|2072
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|60
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|}
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'''Table 1''': The table shows the four different Phusion PCR reactions carried out for the characterisation of the hyperspankoid promoter.
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===Results, Discussion and Conclusion===
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Please refer to:[[Team:Newcastle/7 September 2010#Hyperspankoid_characterisation| 8.09.2010]] for result, discussion and conclusion.
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