Preparation of subtilin immunity BioBrick primers and Phusion PCR

Aims

To rehydrate the subtilin immunity primers. The six primers ordered are Primer 1 P-1, Primer 2 P-2, Primer 1 S-1, Primer 2 S-1, Primer 1 T-1 and Primer 2 T-1. The other primers are Primer 1 V-1 and Primer 2 V-1, which we already have in stock, therefore no rehydration will be needed. (Primer 1 = Forward primer and Primer 2 = Reverse primer).

Phusion PCR will then be performed, using four different DNA fragments, the plasmid vector, promotor & RBS, spaIFEG gene cluster and double terminator.

For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)

Materials and Protocol

Please refer to the DNA Re-hydration protocol.

Please refer to the Phusion PCR protocol.

Table 5: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.

• The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
• Note: The stock primer have to be handled with extra caution, because they will be the main stocks from which working stock solutions will be made from. Therefore, gloves have to be worn to prevent any contamination. Water will have to be from the Pure Lab Distilled Water.

Discussion

Gel electrophoresis for the PCR product will be done on 11 August.

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