(→Gel electrophoresis of subtilin immunity BioBrick fragments)
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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=''rocF'' BioBrick miniprep=
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==Aims==
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The aim of this experiment is to extract pSB1C3 containing the ''rocF'' BioBrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of a nanodrop experiment.
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==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol, [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.
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==Result==
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{|border=1
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|-
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!'''pSB1C3 with ''rocF'' BioBrick (no. 1)'''
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!'''pSB1C3 with ''rocF'' BioBrick (no. 2)'''
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!'''pSB1C3 with ''rocF'' BioBrick (no. 3)'''
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!'''pSB1C3 with ''rocF'' BioBrick (no. 4)'''
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!'''pSB1C3 with ''rocF'' BioBrick (no. 5)'''
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!'''pSB1C3 with ''rocF'' BioBrick (no. 6)'''
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|-
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|90.3 µl/ml
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|64.4 µl/ml
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|286.9 µl/ml
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|87.1 µl/ml
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|780.5 µl/ml
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|59.1 µl/ml
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|}
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'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.
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==Discussion==
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=Gel electrophoresis of subtilin immunity part fragments=
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The tube containing plasmid pSB1C3 with ''rocF'' BioBrick (no.3) has the highest concentration with a high purity value.
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==Conclusion==
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Overall the plasmid miniprep was successful and we would be using Tube no. 3 containing plasmid pSB1C3 with ''rocF'' BioBrick.
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=''rocF'' BioBrick gel electrophoresis=
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==Aims==
==Aims==
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The aim of this experiment is to run gel electrophoresis for the extracted plasmid pSB1C3 containing ''rocF'' BioBrick.
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==Materials and Protocol==
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The aim of this experiment is to do gel extraction for the ''spaIFEG'' gene cluster amplifed on the 12th August, 2010 and the Plasmid Vector, Promoter & RBS and Double terminator amplifed on the 11th August, 2010. Finally, NanoDrop will be performed for all the fragments.
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Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]] for gel electrophoresis protocol.
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==Result==
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[[Image:Newcastle_130810_gel.png|500px]]
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'''Figure 1''': Gel electrophoresis of the miniprep products required for the confirmation of Gibson procedure.
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* '''Lane 1''': 1 kb DNA ladder
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* '''Lane 2''': pSB1C3 with ''rocF'' BioBrick (no. 1)
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* '''Lane 3''': pSB1C3 with ''rocF'' BioBrick (no. 2)
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* '''Lane 4''': pSB1C3 with ''rocF'' BioBrick (no. 3)
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* '''Lane 5''': pSB1C3 with ''rocF'' BioBrick (no. 4)
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* '''Lane 6''': pSB1C3 with ''rocF'' BioBrick (no. 5)
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* '''Lane 7''': pSB1C3 with ''rocF'' BioBrick (no. 6)
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* '''Lane 8''': 1 kb DNA ladder
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==Discussion==
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The expect band size is supposed to be 3195 bp after Gibson procedure ligation. In Figure 1, we can see that in Lane 2 and 5 we got a band of approximately 3200 bp but we found that it is because of the initial insert (''rfp'' gene) which was present in the plasmid pSB1C3 i.e. these 2 sequences are of the template DNA which were used during PCR reaction few days ago. The other 4 bands are of approximately 2100 bp in size.
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==Conclusion==
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Overall the Gibson protocol has failed. We did not receive bands of correct size thus showing that the ''rfp'' fragments and the plasmid pSB1C3 did not ligate at all. The 2 bands of approximately 3200 bp size are of the plasmid pSB1C3 containing ''rfp'' gene insert and thus these fragments are of template DNA for the PCR reaction which had taken place few days ago. The explanation for the failure of the Gibson protocol is as following:
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# Because of the presence of the BioBrick prefix and suffix sequences at the ends of the fragments, the fragments circularized and ligated with themselves. This is because both prefix and suffix contains Not1 restriction site and Xba1 and Spe1 restriction site which have similar sequences and thus during ligation step of the Gibson reaction, the fragments containing prefix and suffix religate with themselves and thus the fragments have not been able to ligate with each other as expected earlier.
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=Subtilin Immunity BioBrick=
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==Aims==
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We plan to select three ''spaIFEG'' gene (part 3) PCR tubes, using Tms 51°C, 56°C and 61°C (we had confirmed it successful from yesterday's gel electrophoresis - please refer to the gel electrophoresis from [[Team:Newcastle/12_August_2010#Results| yesterday]]). Gel extraction will then be performed, along with Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4). Finally, NanoDrop will be performed on each of these four parts.
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==Materials and protocol==
==Materials and protocol==
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Please refer to the [[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]], [[Team:Newcastle/Gel_extraction| gel extraction]] and [[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop]] protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a '''vacuum manifold''' was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out much faster as we had 7 samples.
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Please refer to the [[Team:Newcastle/Gel_extraction| gel extraction]] and [[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop]] protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a '''vacuum manifold''' was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out all at the same time.
[[Image:Newcastle Gel Extraction of spaIFEG genes.JPG|200px|Gel Extraction of spaIFEG genes]]
[[Image:Newcastle Gel Extraction of spaIFEG genes.JPG|200px|Gel Extraction of spaIFEG genes]]
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[[Image:Newcastle Vacuum Manifold 1.JPG|150px|Setting up the tubes for the vacuum manifold]]
[[Image:Newcastle Vacuum Manifold 1.JPG|150px|Setting up the tubes for the vacuum manifold]]
[[Image:Newcastle Vacuum Manifold 2.JPG|150px|Setting up the tubes for the vacuum manifold]]
[[Image:Newcastle Vacuum Manifold 2.JPG|150px|Setting up the tubes for the vacuum manifold]]
[[Image:Newcastle Vacuum Manifold 3.JPG|200px|Vacuum manifold with the QIAquick spin columns loaded with the samples]]
[[Image:Newcastle Vacuum Manifold 3.JPG|200px|Vacuum manifold with the QIAquick spin columns loaded with the samples]]
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[[Image:Newcastle Vacuum Manifold 4.JPG|200px|Vacuum manifold with the QIAquick spin columns empty after the vacuum has been switched on and the samples have passed through]]
==Results==
==Results==
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The results from the Nanodrop Spectrophotometer of the four parts are as follows:
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{|border=1
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*'''Vector''' - 21.3 ng/μl
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*'''Promoter''' - 28.8 ng/μl
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!'''Vector'''
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*'''Coding sequence''' - 27.0 ng/μl
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!'''Promoter'''
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*'''Terminator''' - 27.5 ng/μl
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!'''Immunity gene cluster'''
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!'''Double terminator'''
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|21.3 µl/ml
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|28.8 µl/ml
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|27.0 µl/ml
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|27.5 µl/ml
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|}
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'''Table 1''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
==Discussion==
==Discussion==
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We chose the three ''spaIFEG'' gene (part 3) PCR tubes, using Tms 51°C, 56°C and 61°C because they were the mid-ranged Tms. Gel extraction was then performed, by firstly cutting the gels under U.V light, and then apply the protocol for completion.
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The concentration of the fragments range from 21.3 µl/ml to 28.8 µl/ml. This is acceptable as during the gel extraction step, some of the DNA would be lost.
==Conclusion==
==Conclusion==
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The results from the NanoDrop reading suggests that our results were positive, which indicates that we have successfully extracted all the parts required for the Subtilin Immunity BioBrick. Gibson cloning method will be used next week.
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We have successfully extracted all the parts required for the Subtilin Immunity BioBrick. The Gibson cloning method will be used next week.
Gel electrophoresis of subtilin immunity part fragments
Aims
The aim of this experiment is to do gel extraction for the spaIFEG gene cluster amplifed on the 12th August, 2010 and the Plasmid Vector, Promoter & RBS and Double terminator amplifed on the 11th August, 2010. Finally, NanoDrop will be performed for all the fragments.
Materials and protocol
Please refer to the gel extraction and NanoDrop protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a vacuum manifold was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out all at the same time.
Results
Vector
Promoter
Immunity gene cluster
Double terminator
21.3 µl/ml
28.8 µl/ml
27.0 µl/ml
27.5 µl/ml
Table 1: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
Discussion
The concentration of the fragments range from 21.3 µl/ml to 28.8 µl/ml. This is acceptable as during the gel extraction step, some of the DNA would be lost.
Conclusion
We have successfully extracted all the parts required for the Subtilin Immunity BioBrick. The Gibson cloning method will be used next week.
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