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| - | {| style="color:#1b2c8a;background-color:#3ae2e8;" cellpadding="3" cellspacing="0" border="1" bordercolor="#fff" width="62%" align="center"
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| - | !align="center"|[[Team:Cambridge|Home]]
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| - | !align="center"|[[Team:Cambridge/Team|Team]]
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| - | !align="center"|[https://igem.org/Team.cgi?year=2012&team_name=Cambridge Official Team Profile]
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| - | !align="center"|[[Team:Cambridge/Project|Project]]
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| - | !align="center"|[[Team:Cambridge/Parts|Parts Submitted to the Registry]]
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| - | !align="center"|[[Team:Cambridge/Modeling|Modeling]]
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| - | !align="center"|[[Team:Cambridge/Notebook|Notebook]]
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| - | !align="center"|[[Team:Cambridge/Safety|Safety]]
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| - | !align="center"|[[Team:Cambridge/Attributions|Attributions]]
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| - | !align="center"|[[Team:Cambridge/Sponsors|Sponsors]]
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| - | |}
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| | | | |
| - | {|align="center"
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| - | !align="center"|Week:
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| - | !align="center"|[[Team:Cambridge/Week 1|1]]
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| - | !align="center"|[[Team:Cambridge/Week 2|2]]
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| - | !align="center"|[[Team:Cambridge/Week 3/Lab book|3]]
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| - | !align="center"|[[Team:Cambridge/Week 4/Lab book|4]]
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| - | |}
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| - |
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| - | {|align="center"
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| - | !align="center"|[[Team:Cambridge/Week 4|Diary]]
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| - | !align="center"|[[Team:Cambridge/Week 4/Lab book|Lab book]]
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| - | |}
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| - |
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| - | ===Monday===
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| - | '''Transformation of bacillus and e.coli with lux genes from 2010'''
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| - | ----
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| - | ===Tuesday===
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| - | '''Induction of lux genes in e.coli with IPTG'''
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| - |
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| - | ----
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| - |
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| - | '''[[Team:Cambridge/Protocols/PCRProtocol|PCR of vector]]'''
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| - | ----
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| - | *Plasmid PJS 130 used as our backbone for isolating the magnesium riboswitch.
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| - |
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| - | *Primers:
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| - | :*Forward: TTCAAAACATGACCTATGACgtcgcagagtatgccg
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| - | :*Reverse: cctccctctgctaaaacACAAGGCATTAACACTACAT
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| - |
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| - | *PCR settings:
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| - |
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| - | :* 95 °C - 6mins
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| - |
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| - | ::* 98 °C - 10secs
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| - |
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| - | ::* 58 °C - 45secs
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| - |
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| - | ::* 72 °C - 180secs
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| - |
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| - | :* Repeat above 35x
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| - |
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| - | :* 72 °C - 5mins
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| - | :* 25 °C - 1min
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| - |
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| - | '''[[Team:Cambridge/Protocols/PCRcolony|PCR of riboswitch DNA from ''bacillus'' genome]]'''
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| - |
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| - | ----
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| - |
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| - | * Colony of strain '''check what the strain was''' used as template for this experiment.
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| - |
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| - | * Primers used:
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| - | :* Forward: cggagggagacgattttgTGTTCCGTAATTGTGATGTAAG
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| - |
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| - | :* Reverse: acaccggcatactctgcgacGTCATAGGTCATGTTTTGAACC
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| - | * PCR settings - as above (run in parallel).
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| - | ===Wednesday===
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| - | '''[[Team:Cambridge/Protocols/GelElectrophoresis|Gel electrophoresis of PCR products]]'''
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| - |
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| - | ----
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| - | *PCR fragments for Mg2+ riboswitch from yesterday separated on gel. 90mins at 100V.
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| - |
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| - | [[File:Mg2+ Gel PCR 1.jpg|250px|right|thumb|Results of gel electrophoresis of PCR fragments from yesterday. 550bp fragments in lanes 2+3 were removed before the image was taken]]
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| - | :*Lane 1: Ladder
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| - | :*Lane 2 + 3: Riboswitch DNA from genome (replicates) - 550bp fragment expected.
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| - | :*Lane 4 + 5: Vector DNA from pJS130 (replicates)- Chloramphenicol resistance marker - 9.0kbp fragment expected
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| - |
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| - | :*Lane 6: Postitive control - 1.9kbp fragment expected
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| - |
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| - | *Riboswitch DNA was successfully amplified. However, vector DNA was not. Given that the postive control was also successful, it seems likely that something went wrong with some stage before the PCR amplification. We will try to rectify the problem tomorrow.
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| - |
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| - | '''[[Team:Cambridge/Protocols/GelExtractionofDNA|Purification of riboswitch DNA from gel]]'''
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| - |
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| - | ----
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| - | *Successful riboswitch DNA extracted from gel and purified using a minelute column. DNA frozen for later Gibson assembly.
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| - |
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| - | ===Thursday===
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| - |
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| - | '''[[Team:Cambridge/Protocols/PCRProtocol|PCR of vector]]'''
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| - |
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| - | ----
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| - |
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| - | *Plasmid PJS 130 used as our backbone for isolating the magnesium riboswitch.
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| - |
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| - | *Primers:
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| - |
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| - | :*Forward: TTCAAAACATGACCTATGACgtcgcagagtatgccg
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| - |
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| - | :*Reverse: cctccctctgctaaaacACAAGGCATTAACACTACAT
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| - |
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| - | *PCR settings:
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| - |
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| - | :* 95 °C - 6mins
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| - |
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| - | ::* 98 °C - 10secs
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| - |
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| - | ::* 55 °C - 45secs
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| - |
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| - | ::* 72 °C - 240secs
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| - |
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| - | :* Repeat above 35x
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| - |
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| - | :* 72 °C - 5mins
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| - |
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| - | :* 25 °C - 1min
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| - |
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| - | *Settings changed - Annealing temperature and elongation time - attempting to debug PCR.
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| - |
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| - | '''[[Team:Cambridge/Protocols/GelElectrophoresis|Gel electrophoresis of PCR products]]'''
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| - |
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| - | ----
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| - |
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| - | *PCR fragments for Mg2+ riboswitch from yesterday separated on gel. 90mins at 100V.
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| - |
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| - | [[File:PCR Gel Mg2+ 2.jpg|right|250px|thumb|PCR products from today. Note the unusual band in the negative control and the vector lanes]]
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| - |
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| - | :*Lane 1: Ladder
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| - |
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| - | :*Lane 2, 3 + 4: Vector DNA from pJS130 (replicates) - 9.0kbp fragment expected
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| - |
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| - | :*Lane 5: Postitive control - 1.9kbp fragment expected
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| - | :*Lane 6: Negative control - no fragments expected
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| - | *Once more, the PCR appears to have failed. We will try to redesign the primers tomorrow to make them functional.
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| - |
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| - | ===Friday===
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