(→Overnight cultures for miniprep of filamentous cell part in pGFPrrnB)
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{{Team:Newcastle/mainbanner}}
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=pSB1C3 plasmid gel electrophoresis=
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=Overnight cultures for miniprep of filamentous cell part in pGFP-rrnB=
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==Aims==
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==Aim==
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The aim of this experiment is to run gel electrophoresis for the extracted and linearized plasmid pSB1C3 fragment which were amplified at 4 different melting temperatures by 4 separate PCR reactions.
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The aim of this experiment is to set up cultures for minipreps to obtain stock of the filamentous cell part in pGFP-rrnB for transformation into ''Bacillus subtilis'' 168. We selected 12 colonies for screening.
==Materials and Protocol==
==Materials and Protocol==
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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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Please refer to [[Team:Newcastle/Growing_an_overnight_cultures|growing an overnight culture]].
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
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'''Figure 1''': Gel electrophoresis of plasmid pSB1C3 linearized fragments which are amplified by 4 different PCR reactions running at 4 different melting temperatures.
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* '''Lane 1''': 1 kb DNA ladder
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* '''Lane 2''': pSB1C3 fragment amplified at 55°C
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* '''Lane 3''': pSB1C3 fragment amplified at 55°C
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* '''Lane 4''': pSB1C3 fragment amplified at 60°C
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* '''Lane 5''': pSB1C3 fragment amplified at 60°C
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* '''Lane 6''': pSB1C3 fragment amplified at 65°C
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* '''Lane 7''': pSB1C3 fragment amplified at 65°C
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* '''Lane 8''': pSB1C3 fragment amplified at 70°C
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* '''Lane 9''': pSB1C3 fragment amplified at 70°C
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* '''Lane 10''': 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 sequeich 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.
Overnight cultures for miniprep of filamentous cell part in pGFP-rrnB
Aim
The aim of this experiment is to set up cultures for minipreps to obtain stock of the filamentous cell part in pGFP-rrnB for transformation into Bacillus subtilis 168. We selected 12 colonies for screening.
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