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| - | =pSB1C3 plasmid gel electrophoresis= | + | =Overnight cultures for miniprep of filamentous cell part in pGFP-rrnB= |
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| - | ==Aims== | + | ==Aim== |
| - | 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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| - | ==Materials and Protocol==
| + | 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. |
| - | Please refer to: [[Team:Newcastle/Gel electrophoresis| gel electrophoresis]] for gel electrophoresis protocol.
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| - | | + | |
| - | ==Result==
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| - | [[Image:Newcastle Gel 1 - 23-08-2010.jpg|700px|centre]]
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| - | | + | |
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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==
| + | |
| - | 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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| - | =pSB1C3 plasmid gel electrophoresis by adding EtBr=
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| - | | + | |
| - | ==Aims==
| + | |
| - | 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 and to put Ethidium Bromoide (EtBr) in the agarose gel instead of safeview die so as to get better resolution and brighter bands.
| + | |
| | | | |
| | ==Materials and Protocol== | | ==Materials and Protocol== |
| - | Please refer to: [[Team:Newcastle/Gel electrophoresis| gel electrophoresis]] for gel electrophoresis protocol.
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| | | | |
| - | ==Result==
| + | Please refer to [[Team:Newcastle/Growing_an_overnight_cultures|growing an overnight culture]]. |
| - | [[Image:Newcastle Gel 1 - 23-08-2010.jpg|700px|centre]] | + | |
| | | | |
| | + | '''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.
| |
| | | | |
| - | ==Conclusion==
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| - | The PCR reaction failed as there is no amplification found in any of the reactions.
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| | {{Team:Newcastle/footer}} | | {{Team:Newcastle/footer}} |
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