Amplification of the plasmid PSB1C3 for rocF BioBrick

Aim

The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of rocF BioBrick with the help of a single Phusion PCR using old primers.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the single PCR reaction is mentioned below:

PCR

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 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
• For learning about the rocF fragments, please refer to the Cloning strategy for rocF.

Discussion

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.

Conclusion

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.