Team:Newcastle/4 August 2010

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==Aim==
==Aim==
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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 the Phusion PCR protocol.
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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.
==Materials and Protocol==
==Materials and Protocol==
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!'''Tube'''
!'''Tube'''
!'''Part to be amplified'''
!'''Part to be amplified'''
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!'''DNA fragment consisting the part'''
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!'''Template DNA'''
!'''Forward primer'''
!'''Forward primer'''
!'''Reverse Primer'''
!'''Reverse Primer'''
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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.
'''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 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
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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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* For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
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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'']].
==Discussion==
==Discussion==
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All the 6 Phusion PCR reactions were done however, gel electrophoresis was not done today to check whether the fragments have actually amplified or not.
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The 6 Phusion PCR reactions were carried out.
==Conclusion==
==Conclusion==
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Tomorrow 5th August, 2010, we would be running gel electrophoresis to check the outcome of the 6 PCR reactions and later all the fragments will be ligated with help of Gibson protocol.
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Gel electrophoresis will be carried out tomorrow - 5th August, 2010 to confirm that the ''RocF'' fragments have been successfully amplified.
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{{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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