Team:Newcastle/31 August 2010

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'''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.
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'''Table 1''': Table represents a single Phusion PCR reaction where plasmid pSB1C3 is amplified so that it can be ligated together with other ''rocF'' fragments 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.
* 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 [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
* For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].

Revision as of 10:25, 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 single PCR reaction is 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. 65

Table 1: Table represents a single Phusion PCR reaction where plasmid pSB1C3 is amplified so that it can be ligated together with other rocF fragments 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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