Team:Newcastle/10 August 2010


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Preparation of subtilin immunity BioBrick primers and Phusion PCR


Newcastle 6 primer tubes.JPG

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.

Tube Part to be amplified DNA fragment consisting the part 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 53.3 2046 + 70
2 Promoter and RBS (pVeg-SpoVG) BioBrick Bba_K143053 P1P1 forward P2P2 reverse 51.7 139 + 15
3 spaIFEG Gene Cluster B. subtilis ATCC 6633 P1S1 forward P2S1 reverse 46.0 2753 + 110
4 Double terminator pSB1AK3 consisting BBa_B0014 P1T1 forward P2T1 reverse 50.9 116 + 15

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.


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

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