Team:Newcastle/7 September 2010

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=Hyperspankoid characterisation=
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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.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 02:37, 28 October 2010

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Contents

Hyperspankoid characterisation

Aims

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.

Materials and Protocol

Please refer to Phusion PCR for Phusion PCR protocol.

The details of the four PCR reactions are included in the table below:

Tube Part to be amplified DNA fragment consisting the part i.e. template Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time (in seconds)
1 Hyperspankoid yneA Prom_for HpSpanPspacoid_rev 65 150 15
2 Pspacoid kinA Prom_for HpSpanPspacoid_rev 65 150 15
3 Hyperspank K143055 from Bacillus plate - BS022 Prom_for Pspac-hy rev 62 150 15
4 Plasmid vector pSB1C3/rfp Vector/RFP_for P2-V1 64 2072 60

Table 1: The table shows the four different Phusion PCR reactions carried out for the characterisation of the hyperspankoid promoter.

Results, Discussion and Conclusion

Please refer to: 8.09.2010 for result, discussion and conclusion.


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