Team:Newcastle/4 August 2010

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==Cloning the ''rocF'' BioBrick==
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=Cloning the ''rocF'' BioBrick=
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===Aim===
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==Aim==
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The aim of today's experiment is to construct the ''rocF'' BioBrick and clone it into [http://partsregistry.org/Part:pSB1C3 pSB1C3] using the [[Team:Newcastle/Gibson_Cloning|Gibson assembly method]].
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The aim of this experiment is to amplify 6 different RocF fragments for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] using Phusion PCR.
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==Materials and Protocol==
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Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
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===Phusion PCR===
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{|border=1
 +
|-
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!'''Tube'''
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!'''Part to be amplified'''
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!'''Template DNA'''
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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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|Plasmid Vector
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|pSB1C3
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|P1V1 forward
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|P2V1 reverse
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|58
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|2072 approx.
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|60
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|-
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|2
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|Pspacoid Promoter
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|pMutin4
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|P1P1 forward
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|P2P1 reverse
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|49
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|106 approx.
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|15
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|-
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|3
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|1st fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P1S1 forward
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|P2S1 reverse
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|58
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|246 approx.
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|15
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|-
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|4
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|2nd fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P3S2 forward
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|P4S2 reverse
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|65
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|597 approx.
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|20
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|-
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|5
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|3rd fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P5S3 forward
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|P6S3 reverse
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|66
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|125 approx.
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|15
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|-
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|6
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|Double Terminator
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|pSB1AK3 consisting BBa_B0014 biobrick
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|P1T1 forward
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|P2T1 reverse
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|56
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|116 approx.
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|15
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|}
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'''Table 1''': Table represents 6 different Phusion PCR reactions and the parts which are amplified so that they can be ligated together with the help of Gibson Cloning method.
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* The extension rate of the Phusion polymerase is at 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
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* To learn more about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
 +
 
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==Discussion==
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The 6 Phusion PCR reactions were carried out.
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==Conclusion==
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Gel electrophoresis will be carried out tomorrow - 5th August, 2010 to confirm that the ''RocF'' fragments have been successfully amplified.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 22:49, 27 October 2010

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Contents

Cloning the rocF BioBrick

Aim

The aim of this experiment is to amplify 6 different RocF fragments for the construction of rocF BioBrick using Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:

Phusion PCR

Tube Part to be amplified Template DNA Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time* (in seconds)
1 Plasmid Vector pSB1C3 P1V1 forward P2V1 reverse 58 2072 approx. 60
2 Pspacoid Promoter pMutin4 P1P1 forward P2P1 reverse 49 106 approx. 15
3 1st fragment of rocF CDS B. subtilis 168 chromosome P1S1 forward P2S1 reverse 58 246 approx. 15
4 2nd fragment of rocF CDS B. subtilis 168 chromosome P3S2 forward P4S2 reverse 65 597 approx. 20
5 3rd fragment of rocF CDS B. subtilis 168 chromosome P5S3 forward P6S3 reverse 66 125 approx. 15
6 Double Terminator pSB1AK3 consisting BBa_B0014 biobrick P1T1 forward P2T1 reverse 56 116 approx. 15

Table 1: Table represents 6 different Phusion PCR reactions and the parts which are amplified so that they can be ligated together with the help of Gibson Cloning method.

  • The extension rate of the Phusion polymerase is at 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
  • To learn more about the rocF fragments, please refer to the Cloning strategy for rocF.

Discussion

The 6 Phusion PCR reactions were carried out.

Conclusion

Gel electrophoresis will be carried out tomorrow - 5th August, 2010 to confirm that the RocF fragments have been successfully amplified.

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