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Revision as of 09:37, 31 August 2010

Contents 1 Aim 2 Materials and Protocol 2.1 PCR 3 Discussion 4 Conclusion

Aim

The aim of today's experiment is to amplify 6 different fragments for the construction of rocF BioBrick with the help of 6 different Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:

PCR

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	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 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.