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| - | =Subtilin Immunity and ''rocF''= | + | =Transformation of ligated ''yneA'' into pGFPrrnB and pSB1C3= |
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| - | ==Gel Electrophoresis and Gel Extraction== | + | ==Aims== |
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| - | ===Aims===
| + | To produce more colonies of the plasmid pGFPrrnB and pSB1C3 containing ''yneA'' with pGFPrrnB and pSB1C3 from[[Team:Newcastle/19_August_2010|yesterday]]. |
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| - | Following [[Team:Newcastle/19_August_2010| yesterday]]'s Phusion PCR reactions, we plan to first perform gel electrophoresis of the amplified DNA sequences to check if they are of the correct sizes. If they appear to be successful we will then perform gel extraction, and finally nanodrop to record the level of DNA.
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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]], [[Image:Newcastle Gel 1 (20-08-10).jpg|right|300px|The gel loaded up with the amplified PCR products (blue) and the ladders on either side (green), carried out to check the size of the sequences. Please see results section below for key.]]
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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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| - | [[Image:Newcastle Gel 20-08-2010.jpg|700px|centre]]
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| - | '''Figure 1''': Gel electrophoresis of the amplified PCR products
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| - | *'''Lane 1''': 1 Kb ladder
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| - | *'''Lane 2''': pSB1C3 (for Subtilin Immunity)
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| - | *'''Lane 3''': pSB1C3 (for ''rocF'')
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| - | *'''Lane 4''': pVeg (for Subtilin Immunity)
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| - | *'''Lane 5''': terminator (for Subtilin Immunity)
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| - | *'''Lane 6''': pSpacoid (for ''rocF'')
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| - | *'''Lane 7''': terminator (for ''rocF'')
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| - | *'''Lane 8''': 100 bp ladder
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| - | ===Discussion===
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| - | After looking at the results of our gel, there are no distinct band in lanes 2-3, therefore pSB1C3 vector did not work for both Subtilin Immunity or ''rocF''. However, there are single bands in lanes 4-7 at similar positions to each other. They are about ... in size which is what is expected.
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| - | [[Image:Newcastle Gel 2 (20-08-10) - extraction.jpg|right|300px]]
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| - | [[Image:Newcastle Gel 3 (20-08-10) - extraction.jpg|300px|right]]
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| - | As lanes 2-3 showed no bands, we decided to make a range of Tms to perform another set of PCR reactions to check whether the Tm was the problem. Three Tms were used: 60°C, 65°C and 70°C and six tubes were set up; a duplicate was created for each Tm. After the PCR reactions were finished, there still wasn't any visible pSBIC3 vector band showing up. The problem could have occurred due to the working stocking solution for our primers. Our next step was to re-make the working stock solution for our primers. Also, we had decided to use the Tms 55°C, 60°C, 65°C, 70°C - duplicate for each, making eight PCR tubes in total.
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| - | As lanes 4-7 had bands at the right positions, gel extraction of these four parts was carried out. The concentration of DNA was then measured using the Nanodrop spectrophotometer.
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| - | ===Conclusion===
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| - | The PCR machines were left to run and results of the eight PCR reactions at the 4 different temperatures shall be obtained on [[Team:Newcastle/23_August_2010| Monday]]. After a test gel has been run and if bands appear at the right sizes then gel extraction will be carried out.
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| - | =''yneA''=
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| - | ==Gel Extraction==
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| - | ===Aims===
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| - | To purify the PCR products of digested ''yneA'', pGFPrrnB and pSB1C3 from [[Team:Newcastle/19_August_2010|yesterday]], as well as the promoter and double terminator for the ''rocF'' BioBrick.
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| | ==Materials and Protocol== | | ==Materials and Protocol== |
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| - | Please refer to [[Team:Newcastle/Gel_extraction|gel extraction]] and [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop]].
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| - | ===Results===
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| - | The table below shows results of concentration (in ng/µl) from NanoDrop:
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| - | {|border=1
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| - | ! Sample no.
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| - | ! ''yneA''
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| - | ! pGFPrrnB
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| - | ! pSB1C3
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| - | |-
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| - | !1
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| - | !432.5
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| - | !238.9
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| - | !126.1
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| - | |-
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| - | !2
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| - | !347.6
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| - | !230.7
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| - | !107.6
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| - | |-
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| - | !3
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| - | !380.2
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| - | !390.9
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| - | !121.5
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| - | |-
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| - | !4
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| - | !377.9
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| - | !236.2
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| - | !112.8
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| - | |}
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| - | ===Discussion===
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| - | Results from the NanoDrop show concentration values of more than 100ng/µl, which means we have obtained good concentration of DNA from the miniprep.
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| - | ===Conclusion===
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| - | ==Miniprep==
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| - | ===Aim===
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| - | To produce stocks of ''yneA'', pGFPrrnB and pSB1C3.
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| - | ===Materials and Protocol===
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| - | Please refer to [[Team:Newcastle/Qiagen_Minipreps|miniprep]] and [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop]].
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| - | ===Results===
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| - | ===Discussion===
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| - | ===Conclusion===
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| - | ==Transformation of Ligated Products==
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| - | ===Aims===
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| - | To produce more colonies of the ligated products of ''yneA'' with pGFPrrnB and pSB1C3.
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| - | ===Materials and Protocol===
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| | Please refer to [[Team:Newcastle/Transformation_of_E._coli|transformation of E. coli]]. | | Please refer to [[Team:Newcastle/Transformation_of_E._coli|transformation of E. coli]]. |
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| - | ==Ligation==
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| - | ===Aims===
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| - | To ligate ''yneA'' with pGFPrrnB and ''yneA'' with pSB1C3 (A repeat of [[Team:Newcastle/19_August_2010|yesterday]].
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| - | ===Materials and Protocol===
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| - | Please refer to [[Team:Newcastle/Ligation|ligation]].
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| - | ===Results, Discussion and Conclusion===
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| - | Please refer to [[Team:Newcastle/23_August_2010|23.08.10]].
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| - | ==Plating ''Bacillus subtilis'' BFS678==
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| - | Has pMutin4 (and thus ''lacI'') integrated into the chromosome...will be useful for characterisation.. will be transforming this strain with our filamentous cell and arginase BioBricks.
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| | {{Team:Newcastle/footer}} | | {{Team:Newcastle/footer}} |
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