Team:Newcastle/6 August 2010

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(New page: =Gel Electrophoresis for the Amplified Fragments of ''rocF''= ==Aim== The aim of the experiment is to perform gel electrophoresis for all the 6 PCR reactions which took place yesterday 4t...)
 
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=Gel Electrophoresis for the Amplified Fragments of ''rocF''=
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{{Team:Newcastle/mainbanner}}
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=Gel Electrophoresis for the amplified fragments of Pspac_oid promoter and ''lacI''=
==Aim==
==Aim==
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The aim of the experiment is to perform gel electrophoresis for all the 6 PCR reactions which took place yesterday 4th August, 2010 and thus confirm that all 6 PCR reactions were successful.
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The aim of the experiment is to perform gel electrophoresis for the two PCR reactions, ''lacI'' and Pspac_oid promoter that were done using a different stock of plasmid pMutin4 on 5th August, 2010.
==Materials and Protocol==
==Materials and Protocol==
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==Result==
==Result==
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[[Image:Newcastle_060810_gel_1.png|400px]]
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'''Figure 1''': Gel electrophoresis of the ''lacI'' and  Pspac_oid promoter.
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* '''Lane 1''': 1kb DNA ladder
* '''Lane 1''': 1kb DNA ladder
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* '''Lane 2''': BioBrick compatible vector pSB1C3
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* '''Lane 2''': Plamid pMutin4 containing ''lacI''
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* '''Lane 3''': Pspac_oid promoter
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* '''Lane 3''': Plamid pMutin4 containing Pspac_oid promoter
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* '''Lane 4''': 1st fragment of ''rocF'' CDS
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* '''Lane 4''': 100bp DNA ladder
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* '''Lane 5''': 2nd fragment of ''rocF'' CDS
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* '''Lane 6''': 3rd fragment of ''rocF'' CDS
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* '''Lane 7''': Double Terminator
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* '''Lane 8''': 1kb DNA ladder
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{|border=1
{|border=1
|-
|-
!
!
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!'''Biobrick compatible vector pSB1C3'''
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!'''Pspac_oid promoter'''
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!'''Pspac_oid pormoter'''
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!'''''lacI'''''
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!'''1st fragment of ''rocF'' CDS'''
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!'''2nd fragment of ''rocF'' CDS)'''
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!'''3rd fragment of ''rocF'' CDS'''
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!'''Double Terminator'''
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|-
|-
|'''Size of the Fragment (in bp)'''
|'''Size of the Fragment (in bp)'''
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|2072 approx.
 
|106 approx.
|106 approx.
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|246 approx.
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|1400 approx.
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|597 approx.
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|125 approx.
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|116 approx.
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|}
|}
'''Table 1''': Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
'''Table 1''': Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
==Discussion==
==Discussion==
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We found bands in the lanes 2,4,5,6 and 7 of the correct sizes but lane 3 did not contain any band.
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A correct sized band was observed in lane 2, ''lacI'' which serve as the positive control. However no band was observed in lane 3, which contain the Pspac_oid promoter.
==Conclusion==
==Conclusion==
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This experiment shows that the PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was present in the lane 3 and did not show any band. This could be because of the following problems:
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This experiment shows that the plasmid pMutin4 is intact as the ''lacI'' frangment have been successfully amplified (Lane 2). However the Pspac_oid promoter are still not being amplified. This could be due to the following reason:
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# Primer sequences could be incorrect.
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# Melting temperature could be incorrect.
# Melting temperature could be incorrect.
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# Plasmid pMutin4 could have degenerated due to long term storage.
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 +
==Solution for the problem==
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# Try a range of melting temperature.
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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 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 2 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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|49
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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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|50
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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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==Result==
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[[Image:Newcastle_060810_gel_2.png|400px]]
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'''Figure 2''': Gel electrophoresis of the Pspac_oid promoter from plasmid pMutin4.
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* '''Lane 1''': 100bp DNA ladder
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* '''Lane 2''': Plamid pMutin4 containing Pspac_oid promoter at 49°C
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* '''Lane 3''': Plamid pMutin4 containing Pspac_oid promoter at 50°C
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* '''Lane 4''': 100bp DNA ladder
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 +
{|border=1
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|-
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!
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!'''Pspac_oid promoter at 49°C'''
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!'''Pspac_oid promoter at 50°C'''
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|-
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|'''Size of the Fragment (in bp)'''
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|106 approx.
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|106 approx.
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|}
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'''Table 3''': Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.
 +
 
 +
==Discussion==
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We found two very faint bands in the lanes 2 and 3.
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==Conclusion==
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This experiment shows that the PCR reaction was unsuccessful even though we got a faint band for Pspac_oid promoter. This time, we are going to use plasmid containing KinA Biobrick and plasmid containing stochastic switch Biobrick which were devised by Team Newcastle 2009. We would be dealing with this on Monday 9th August, 2010.
 +
 
 +
{{Team:Newcastle/footer}}

Latest revision as of 23:24, 25 October 2010

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Contents

Gel Electrophoresis for the amplified fragments of Pspac_oid promoter and lacI

Aim

The aim of the experiment is to perform gel electrophoresis for the two PCR reactions, lacI and Pspac_oid promoter that were done using a different stock of plasmid pMutin4 on 5th August, 2010.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

Newcastle 060810 gel 1.png

Figure 1: Gel electrophoresis of the lacI and Pspac_oid promoter.

  • Lane 1: 1kb DNA ladder
  • Lane 2: Plamid pMutin4 containing lacI
  • Lane 3: Plamid pMutin4 containing Pspac_oid promoter
  • Lane 4: 100bp DNA ladder
Pspac_oid promoter lacI
Size of the Fragment (in bp) 106 approx. 1400 approx.

Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

A correct sized band was observed in lane 2, lacI which serve as the positive control. However no band was observed in lane 3, which contain the Pspac_oid promoter.

Conclusion

This experiment shows that the plasmid pMutin4 is intact as the lacI frangment have been successfully amplified (Lane 2). However the Pspac_oid promoter are still not being amplified. This could be due to the following reason:

  1. Melting temperature could be incorrect.

Solution for the problem

  1. Try a range of melting temperature.

Amplification of Pspac_oid promoter by PCR

Aim

The aim of this experiment is to amplify the Pspac_oid promoter from plasmid pMutin4 for the construction of rocF BioBrick using Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 2 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 Pspacoid Promoter pMutin4 P1P1 forward P2P1 reverse 49 106 approx. 15
2 Pspacoid Promoter pMutin4 P1P1 forward P2P1 reverse 50 106 approx. 15

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.

  • The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
  • To learn more about the rocF fragments, please refer to the Cloning strategy for rocF.

Result

Newcastle 060810 gel 2.png

Figure 2: Gel electrophoresis of the Pspac_oid promoter from plasmid pMutin4.

  • Lane 1: 100bp DNA ladder
  • Lane 2: Plamid pMutin4 containing Pspac_oid promoter at 49°C
  • Lane 3: Plamid pMutin4 containing Pspac_oid promoter at 50°C
  • Lane 4: 100bp DNA ladder
Pspac_oid promoter at 49°C Pspac_oid promoter at 50°C
Size of the Fragment (in bp) 106 approx. 106 approx.

Table 3: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

We found two very faint bands in the lanes 2 and 3.

Conclusion

This experiment shows that the PCR reaction was unsuccessful even though we got a faint band for Pspac_oid promoter. This time, we are going to use plasmid containing KinA Biobrick and plasmid containing stochastic switch Biobrick which were devised by Team Newcastle 2009. We would be dealing with this on Monday 9th August, 2010.

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