Team:Newcastle/31 August 2010

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(Cloning the rocF BioBrick)
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==Aim==
==Aim==
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The aim of today's experiment is to amplify 6 different fragments for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 6 different Phusion PCR.
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The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of a single Phusion PCR using old primers.
==Materials and Protocol==
==Materials and Protocol==

Revision as of 09:47, 31 August 2010

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Contents

Amplification of the plasmid PSB1C3 for rocF BioBrick

Aim

The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of rocF BioBrick with the help of a single Phusion PCR using old primers.

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.



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