Team:Newcastle/4 August 2010

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|58
|58
|246 approx.
|246 approx.
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|15
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|-
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|4
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|2nd fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P3S2 forward
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|P4S2 reverse
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|65
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|597 approx.
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|20
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|5
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|3rd fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P5S3 forward
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|P6S3 reverse
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|66
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|125 approx.
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|15
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|6
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|Double Terminator
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|pSB1AK3 consisting BBa_B0014 biobrick
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|P1T1 forward
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|P2T1 reverse
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|116 approx.
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Revision as of 09:27, 5 August 2010

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Contents

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

Gibson assembly

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