From 2010.igem.org

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.

Team:Newcastle/4 August 2010