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| - | =Gel exlectrophoresis for single digestion of pSB1C3=
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| - | ==Aim==
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| - | The aim of this experiment is to check if the digestion from [[Team:Newcastle/24_August_2010|yesterday]] worked.
| + | =Transformation of ''Bacillius subtilis'' 168 with pGFP-rrnB containing ''yneA''= |
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| - | ==Materials and Protocol== | + | ==Aim== |
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| - | Please refer to [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]] for protocol.
| + | We performed gel electrophoresis on our 12 digested minipreps and found 8 were the correct product. We transformed ''B. subtilis'' 168 with one of the samples (1). |
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| - | ==Results==
| + | [[Image:filamentous_in_pgfprrnb.jpg|800px]] |
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| - | The result from gel electrophoresis:
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| - | [[Image:Newcastle 25-08-2010 – Gel 1 (Ethidium Bromide).png|500px|centre]] | + | |
| - | '''Figure 1''' shows the double digest of 12 tubes of pGFPrrnB and ''yneA''.
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| - | *'''Lane 1''': Vector only
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| - | *'''Lane 2''': Tube 1
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| - | *'''Lane 3''': Tube 2
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| - | *'''Lane 4''': Tube 3
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| - | *'''Lane 5''': Tube 4
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| - | *'''Lane 6''': Tube 5
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| - | *'''Lane 7''': Tube 6
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| - | *'''Lane 8''': Tube 7
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| - | *'''Lane 9''': Tube 8
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| - | *'''Lane 10''': Tube 9
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| - | *'''Lane 11''': Tube 10
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| - | *'''Lane 12''': Tube 11
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| - | *'''Lane 13''': Tube 12
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| - | ===Discussion===
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| - | The bands we got from the gel shows that digestion in tubes 1, 2, 4, 10, 11 and 12 worked.
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| - | ===Conclusion===
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| - | We use the digested products from the six tubes that worked for ligation.
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| - | ==Overnight culture for transformation of ''B. subtilis'' with ''yneA''==
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| - | ==Aim==
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| - | To transform ''yneA'' into competent ''B. subtilis''.
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| | ==Materials and Protocol== | | ==Materials and Protocol== |
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| | Please refer to results in [[Team:Newcastle/26_August_2010|tomorrow]]'s lab book. | | Please refer to results in [[Team:Newcastle/26_August_2010|tomorrow]]'s lab book. |
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| - | ==Transformation==
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| - | ===Aim===
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| - | To transform pGFPrrnB and ''yneA'' into E. coli.
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| - | ===Materials and Protocol===
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| - | Please refer to [[Team:Newcastle/Transformation_of_E._coli|transformation of E. coli]].
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| - | ===Results and Conclusion===
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| - | Please refer to [[Team:Newcastle/26_August_2010|tomorrow]]'s lab book.
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| - | ==PCR==
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| - | ===Aim===
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| - | To amplify the DNA pSB1C3 using RocF primer.
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| - | ===Materials and Protocol===
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| - | Please refer to [[Team:Newcastle/PCR|PCR]].
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| - | ===Conclusion===
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| - | Continue with PCR purification.
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| - | ==PCR Purification==
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| - | ===Aim===
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| - | To remove unwanted primers, taq polymerase, buffer and salts to obtain pure DNA.
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| - | ===Materials and Protocol===
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| - | Please refer to [[Team:Newcastle/PCR_purification|PCR purification]].
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| - | ==Digestion==
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| - | ===Aim===
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| - | To digest the PCR products of pSB1C3 and ''yneA'' from PCR purification.
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| - | ===Materials and Protocol===
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| - | Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
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| - | ==Gel extraction==
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| - | ===Aim===
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| - | To purify the DNA of ''yneA'' and pSB1C3 by extracting the bands from the gel after running gel electrophoresis. Concentration of DNA is then checked with NanoDrop.
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| - | ===Materials and Protocol===
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| - | Please refer to:
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| - | * [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]],
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| - | * [[Team:Newcastle/Gel_extraction|gel extraction]] and
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| - | * [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]].
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| - | ===Results===
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| - | The bands we got from gel electrophoresis is very faint.
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| - | ===Conclusion===
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| - | We realized that we used the wrong ''rocF'' primer, so we repeat the whole procedure from PCR again.
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| - | =Restriction digestion and gel extraction linearized pSB1C3=
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| - | ==Aims==
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| - | The aim of this experiment is to digested the plasmid pSB1C3 with the restriction enzyme HindIII to linearize it and and to perform gel extraction to purify it.
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| - | ==Materials and protocol==
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| - | Please refer to the:
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| - | *[[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]],
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| - | *[[Team:Newcastle/Gel_extraction| gel extraction]] and
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| - | *[[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop spectrophotometer]] protocols.
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| - | ==Results==
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| - | *'''Lane 1''': 1 Kb ladder
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| - | *'''Lane 2''': Linearized plasmid pSB1C3
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| - | *'''Lane 3''': 1 Kb ladder
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| - | There is no gel photograph because we want to keep the exposure of DNA to the UV light to an absolute minimum.
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| - | ==Discussion==
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| - | During gel extraction procedure, we found a bright band of approx
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| - | During gel extraction procedure, we found a bright band of approximately 3100 bp size in lane 2 under UV light and we cut the gel and extracted the band.
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| - | ==Conclusion==
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| - | We got linearized plasmid pSB1C3 and we performed gel extraction successfully and the nanodrop protocol showed that we got 12.7 ng/µl concentration of plasmid.
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| | {{Team:Newcastle/footer}} | | {{Team:Newcastle/footer}} |
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