"
Team:Newcastle/9 August 2010
From 2010.igem.org
| |||||||||||||
| |||||||||||||
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 | 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 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 pMK-RQ containing Biobrick kinA and plasmid pMK-RQ 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 | 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 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 4 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 4 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
Gel Electrophoresis for Amplified Pspac_oid promoter and lacI
Aim
The aim of the experiment is to perform gel electrophoresis for the two PCR reactions viz. lacI and Pspac_oid promoter which took place yesterday 5th August, 2010 and thus confirm that they were successful.
Materials and Protocol
Please refer to: Gel electrophoresis.
Result
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 pormoter | 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
We found a band in the lanes 2 of the correct size but lane 3 did not contain any band.
Conclusion
This experiment shows that the PCR reaction was successful for the lacI fragment apart from Pspac_oid promoter which was present in the lane 3 and did not show any band. As we got a band for lacI, we can conclude that the plasmid pMutin4 is intact. But we still are not getting a band for Pspac_oid pomoter. This could be because of the following problem:
- Melting temperature could be incorrect.
Transformation of hyperspank and spoVG
Aim
To transform competent E.coli DH5α with hyperspank and spoVG.
Materials and Protocol
Please refer to transformation of E.coli.
   
|
    
|
