Team:Newcastle/23 August 2010

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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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[[Image:Newcastle Gel 1 - 23-08-2010.jpg|700px|centre]]
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
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'''Figure 1''': Gel electrophoresis of the amplified linearized plasmid pSB1C3 fragments ran at 4 different melting temperatures, Tms, (50, 60, 65, 70°C). A 1 kb DNA ladder was used on either side of lanes.
 
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* '''Lane 1''': pSB1C3 fragment amplified at 55°C
 
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* '''Lane 2''': pSB1C3 fragment amplified at 60°C
 
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* '''Lane 3''': pSB1C3 fragment amplified at 65°C
 
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* '''Lane 4''': pSB1C3 fragment amplified at 70°C
 
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* '''Lane 5''': pSB1C3 fragment amplified at 55°C
 
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* '''Lane 6''': pSB1C3 fragment amplified at 60°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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==Discussion==
 
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No bands were found in any of the lanes. Yesterday, a faint band was found when the melting temperature was set at 65°C but today no band is found in lane 3 and lane 7. This makes finding the cause for no amplification even difficult. We would still be looking into it and would be changing other parameters.
 
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==Conclusion==
 
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The PCR reaction failed as there is no amplification found in any of the reactions.
 
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Latest revision as of 00:44, 28 October 2010

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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.

Materials and Protocol

Please refer to growing an overnight culture.

Go back to our main Lab book page



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