Team:Newcastle/9 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Amplification of Pspac_oid promoter by PCR=
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=Subtilin immunity part=
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==Aim==
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Today we were working on our subtilin immunity part cloning strategy.
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The aim of this experiment is to amplify the Pspac_oid promoter fragment from plasmid pMutin4 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] using Phusion PCR.
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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 4 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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|Pspacoid Promoter
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|pMutin4
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|P1P1 forward
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|P2P1 reverse
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|58
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|106 approx.
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|15
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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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|59
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|106 approx.
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|15
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|}
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'''Table 1''': Table represents 4 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of ''rocF'' with the help of Gibson Cloning method.
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* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
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* To learn more 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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For the gel electrophoresis results, please refer to the bottom section.
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=Amplification of Pspac_oid promoter by PCR=
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==Aim==
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The aim of this experiment is to amplify the Pspac_oid promoter fragment from plasmid pMK-RQ containing Biobrick ''kinA'' and plasmid pMK-RQ containing stochastic switch developed by Team Newcastle 2009 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] using Phusion PCR.
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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 2 PCR reactions are mentioned below:
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===PCR===
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-
 
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{|border=1
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-
|-
+
-
!'''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)'''
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-
!'''Extension time* (in seconds)'''
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-
|-
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|1
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|Pspacoid Promoter
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|Plasmid containing ''kinA''
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|P1P1 forward
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|P2P1 reverse
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|58
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|106 approx.
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|15
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|-
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|2
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|Pspacoid Promoter
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|Plasmid containing ''kinA''
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|P1P1 forward
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|P2P1 reverse
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|59
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|106 approx.
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|15
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|-
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|3
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|Pspacoid Promoter
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|Plasmid containing stochastic switch
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|P1P1 forward
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|P2P1 reverse
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|58
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|106 approx.
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|15
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|-
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|1
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|Pspacoid Promoter
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|Plasmid containing stochastic switch
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|P1P1 forward
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|P2P1 reverse
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|59
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|106 approx.
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|15
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|}
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'''Table 2''': Table represents 2 different Phusion PCR reactions for the amplification of Pspac_oid promoter, so that it can be ligated together with other fragments for the construction of ''rocF'' with the help of Gibson Cloning method.
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* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
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* To learn more 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 4 Phusion PCR reactions were done however, gel electrophoresis will be done later today, to check whether the fragments have actually amplified or not.
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==Conclusion==
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Today afternoon, we would be running gel electrophoresis to check the outcome of the 4 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
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=Gel Electrophoresis for Amplified Pspac_oid promoter=
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==Aim==
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The aim of the experiment is to perform gel electrophoresis for the two PCR reactions Pspac_oid promoter amplified from plasmid pMutin4 and from plasmid pMK-RQ containing Biobrick ''kinA'' and plasmid pMK-RQ containing stochastic switch developed by Team Newcastle 2009 which took place today morning and thus confirm that they were successful.
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==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
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==Result==
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[[Image:Newcastle_090810_gel_2.png|400px]]
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'''Figure 1''': Gel electrophoresis of the ''lacI'' and  Pspac_oid promoter.
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* '''Lane 1''': 100bp DNA ladder
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* '''Lane 2''': Plamid pMutin4 containing Pspac_oid promoter
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* '''Lane 3''': Plamid pMutin4 containing Pspac_oid promoter
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* '''Lane 4''': Plamid pMK-RQ (''kinA'' BioBrick) containing Pspac_oid promoter
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* '''Lane 5''': Plamid pMK-RQ (''kinA'' BioBrick) containing Pspac_oid promoter
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* '''Lane 6''': Plamid pMK-RO (stchastic switch BioBrick) containing Pspac_oid promoter
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* '''Lane 7''': Plamid pMK-RO (stchastic switch BioBrick) containing Pspac_oid promoter
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* '''Lane 8''': 100bp DNA ladder
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{|border=1
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|-
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!
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!'''Pspac_oid pormoter'''
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|-
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|'''Size of the Fragment (in bp)'''
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|148 approx.
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|}
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'''Table 3''': Table represents the size of the Pspac_oid fragment represented as bands on the gel in all the lanes.
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==Discussion==
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We found two bands in the all lanes out of which one is of approximately of 150 bp is size and the other band is of 80 bp approximately in size.
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==Conclusion==
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This experiment shows that the PCR reaction was not perfectly successful for the Pspac_oid fragment. The problem is that there are 2 fragments on the gel instead of 1 and the second band which of 80bp in size might be corresponding to primer dimers. When we checked the primer sequence again, we found that there is a high complementarity within the primers itself and thus the amplification of the Pspac_oid fragment is not taking place at a desired rate. The first band present on the gel can be of the Pspac_oid fragment and thus tomorrow 10th August, 2010, we would be doing gel extraction for the fragment and will check whether these fragments are of Pspac_oid or not.
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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 23:03, 27 October 2010

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Today we were working on our subtilin immunity part cloning strategy.

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