Team:Newcastle/4 August 2010

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==Aim==
==Aim==
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The aim of today's experiment is to amplify 6 different fragments for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of 6 different Phusion PCR.
+
The aim of this experiment is to amplify 6 different RocF fragments for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] using Phusion PCR.
==Materials and Protocol==
==Materials and Protocol==
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
Please refer to  [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
-
===PCR===
+
===Phusion PCR===
{|border=1
{|border=1
|-
|-
-
!'''pSB1C3
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!'''Tube'''
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(No. 1)'''
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!'''Part to be amplified'''
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!'''pSB1C3
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!'''Template DNA'''
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(No. 2)'''
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!'''Forward primer'''
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!'''pSB1C3
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!'''Reverse Primer'''
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(No. 3)'''
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!'''Melting Temperature (Tm in °C) '''
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!'''pSB1C3
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!'''Size of the fragment (in bp)'''
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(No. 4)'''
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!'''Extension time* (in seconds)'''
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!'''lacI
+
-
(No. 1)'''
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-
!'''lacI
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-
(No. 2)'''
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!'''Double terminator !'''Double terminator
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-
(No. 1)'''
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-
!'''Double terminator
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-
(No. 2)'''
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|-
|-
-
|44.0 µl/ml
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|1
-
|19.9 µl/ml
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|Plasmid Vector
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|25.0 µl/ml
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|pSB1C3
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|30.8 µl/ml
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|P1V1 forward
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|10.0 µl/ml
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|P2V1 reverse
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|44.2 µl/ml
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|58
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|9.2 µl/ml
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|2072 approx.
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|39.7 µl/ml
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|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==
+
'''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.
 +
* The extension rate of the Phusion polymerase is at 1Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
 +
* To learn more about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']].
 +
 
 +
==Discussion==
 +
The 6 Phusion PCR reactions were carried out.
 +
 
 +
==Conclusion==
 +
Gel electrophoresis will be carried out tomorrow - 5th August, 2010 to confirm that the ''RocF'' fragments have been successfully amplified.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 22:49, 27 October 2010

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Contents

Cloning the rocF BioBrick

Aim

The aim of this experiment is to amplify 6 different RocF fragments for the construction of rocF BioBrick using Phusion PCR.

Materials and Protocol

Please refer to PCR for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:

Phusion PCR

Tube Part to be amplified Template DNA 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

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.

  • The extension rate of the Phusion polymerase is at 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.

Discussion

The 6 Phusion PCR reactions were carried out.

Conclusion

Gel electrophoresis will be carried out tomorrow - 5th August, 2010 to confirm that the RocF fragments have been successfully amplified.

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