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Team:Newcastle/9 August 2010
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==Materials and Protocol== | ==Materials and Protocol== | ||
| - | Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the | + | Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. The details for the 4 PCR reactions are mentioned below: |
===PCR=== | ===PCR=== | ||
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|1 | |1 | ||
|Pspacoid Promoter | |Pspacoid Promoter | ||
| - | | | + | |Plasmid containing ''kinA'' |
|P1P1 forward | |P1P1 forward | ||
|P2P1 reverse | |P2P1 reverse | ||
| Line 62: | Line 62: | ||
|2 | |2 | ||
|Pspacoid Promoter | |Pspacoid Promoter | ||
| - | | | + | |Plasmid containing ''kinA'' |
| + | |P1P1 forward | ||
| + | |P2P1 reverse | ||
| + | |59 | ||
| + | |106 approx. | ||
| + | |15 | ||
| + | |- | ||
| + | |3 | ||
| + | |Pspacoid Promoter | ||
| + | |Plasmid containing stochastic switch | ||
| + | |P1P1 forward | ||
| + | |P2P1 reverse | ||
| + | |58 | ||
| + | |106 approx. | ||
| + | |15 | ||
| + | |- | ||
| + | |1 | ||
| + | |Pspacoid Promoter | ||
| + | |Plasmid containing stochastic switch | ||
|P1P1 forward | |P1P1 forward | ||
|P2P1 reverse | |P2P1 reverse | ||
| Line 70: | Line 88: | ||
|} | |} | ||
| - | '''Table | + | '''Table 2''': 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. |
* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different. | * The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different. | ||
* For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']]. | * For learning about the ''rocF'' fragments, please refer to the [[Media:Cloning_strategy_for_rocF.pdf|Cloning strategy for ''rocF'']]. | ||
Revision as of 13:53, 9 August 2010
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Contents |
Transformation of pVeg and lacI
Results/Aims
- There are colonies on all 3 plates for pVeg.
- For lacI, there is only one colony on one of the normal plates and more on the concentrated one.
Discussion
- For pVeg, we will set up an overnight broth culture from the colonies for plasmid extraction
- To show that we have sucessfully transformed from lacI, we will select 4 colonies and plate them on a section of an agar plate as well as setting up overnight broth cultures.
Conclusion
Please refer to 10.08.10.
Transformation of lacI
Aims
To transform some competent E.coli DH5α with the ligation products: 1:3, 1:5 and the vector from the ligation on 06.08.10.
Materials and Protocol
Please refer to Transformation of E.coli.
Result
Please refer to 10.08.10 for result.
Amplification of Pspac_oid promoter by PCR
Aim
The aim of this experiment is to amplify Pspac_oid promoter fragment from plasmid pMutin4 for the construction of rocF BioBrick with the help of 2 different Phusion PCR.
Materials and Protocol
Please refer to PCR for Phusion PCR protocol. The details for the 4 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 | Plasmid containing kinA | P1P1 forward | P2P1 reverse | 58 | 106 approx. | 15 |
| 2 | Pspacoid Promoter | Plasmid containing kinA | P1P1 forward | P2P1 reverse | 59 | 106 approx. | 15 |
| 3 | Pspacoid Promoter | Plasmid containing stochastic switch | P1P1 forward | P2P1 reverse | 58 | 106 approx. | 15 |
| 1 | Pspacoid Promoter | Plasmid containing stochastic switch | P1P1 forward | P2P1 reverse | 59 | 106 approx. | 15 |
Table 2: 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.
- The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
- For learning about the rocF fragments, please refer to the Cloning strategy for rocF.
Discussion
All the 2 Phusion PCR reactions were done however, gel electrophoresis will be done later today, to check whether the fragments have actually amplified or not.
Conclusion
Today afternoon, we would be running gel electrophoresis to check the outcome of the 2 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
Amplification of Pspac_oid promoter by PCR
Aim
The aim of this experiment is to amplify Pspac_oid promoter fragment from plasmid containing Biobrick kinA and plasmid containing stochastic switch developed by Team Newcastle 2009 for the construction of rocF BioBrick with the help of 4 different 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 | 58 | 106 approx. | 15 |
| 2 | Pspacoid Promoter | pMutin4 | P1P1 forward | P2P1 reverse | 59 | 106 approx. | 15 |
Table 1: 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. Thus the extension time of each and every PCR reaction is slightly different.
- For learning about the rocF fragments, please refer to the Cloning strategy for rocF.
Discussion
All the 2 Phusion PCR reactions were done however, gel electrophoresis will be done later today, to check whether the fragments have actually amplified or not.
Conclusion
Today afternoon, we would be running gel electrophoresis to check the outcome of the 2 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
   
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