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Contents 1 Cloning the rocF BioBrick 1.1 Aim 1.2 Materials and Protocol 1.2.1 PCR 1.3 Gibson assembly

Cloning the rocF BioBrick

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	Plasmid 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.

Gibson assembly