Team:Newcastle/31 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Amplification of the plasmid PSB1C3 for ''rocF'' BioBrick=
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=Glycerol stocks=
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Today we made [[Team:Newcastle/Glycerol_stocks|glycerol stocks]] of our filamentous ''Bacillus subtilis'' 168 strain.
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==Aim==
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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.
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==Materials and Protocol==
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Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
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===PCR===
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{|border=1
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|-
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!'''Tube'''
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!'''Part to be amplified'''
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!'''DNA fragment consisting the part'''
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!'''Forward primer'''
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!'''Reverse Primer'''
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!'''Melting Temperature (Tm in °C) '''
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!'''Size of the fragment (in bp)'''
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!'''Extension time* (in seconds)'''
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|-
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|1
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|Plasmid Vector
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|pSB1C3
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|P1V1 forward
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|P2V1 reverse
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|58
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|2072 approx.
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|60
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|-
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|2
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|Pspacoid Promoter
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|pMutin4
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|P1P1 forward
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|P2P1 reverse
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|49
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|106 approx.
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|15
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|-
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|3
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|1st fragment of ''rocF'' CDS
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|''B. subtilis'' 168 chromosome
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|P1S1 forward
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|P2S1 reverse
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|58
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|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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|-
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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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|-
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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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|56
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|116 approx.
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|15
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|}
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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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* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
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* For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
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==Discussion==
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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.
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==Conclusion==
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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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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 01:20, 28 October 2010

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Glycerol stocks

Today we made glycerol stocks of our filamentous Bacillus subtilis 168 strain.

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