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Team:Newcastle/4 August 2010
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==Aim== | ==Aim== | ||
| - | The aim of this experiment is to amplify 6 different RocF fragments for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] using Phusion PCR . | + | 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== | ||
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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