Team:Newcastle/4 August 2010


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Cloning the rocF BioBrick


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.


The 6 Phusion PCR reactions were carried out.


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

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