Team:Freiburg/Notebook/12 July

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<a href="https://2011.igem.org/Team:Freiburg/Notebook/11_July">Previous entry</a>
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 12 July </a>
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=Sequence Analysis=
=Sequence Analysis=
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==S15b==
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==<span style="color:orange;">Lysis cassette</span>==  
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Lysis cassette K124014, named as K124017  
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===Lysis cassette and heat inducible promotor===
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S15b: (Lysis cassette K124014) is named K124017 in the registry
Part without RBS at the beginning
Part without RBS at the beginning
Part without antiholin
Part without antiholin
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the sequece is also mutated leading to wrong amino acids
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if combined with RBS there would be too many bases (8) between RBS and coding sequence
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part is likely to be not effective
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==<span style="color:green;">green light receptor</span>==
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===CcaR===
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S17: (CcaR with Prefix and RBS) is ok. Check RBS.
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S18: seems to be ok too.
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==<span style="color:red;">red light receptor</span>==
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===pycA+terminator===
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S24: prefix and suffix not complete
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===cph8===
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S29: Sequence without part. Has to be done again.
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'''Digestion'''
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{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name:
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date:
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|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Date Name
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Experiment
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|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
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|}
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Procedure
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# add H<sub>2</sub>O (38μl-DNA )
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# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
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# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
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# DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
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# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
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# heat for 1-2 hours 37°C (6 hours if time)
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# heat for 20 minutes 80°C (inactivation of enzymes)
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# keep at 4°C if you cannot continue
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Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
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{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Sample Name
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA concentration (μg/μl)
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P20 GFP
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 132
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P19 GFP
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 145
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P18 GFP
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 107
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S39 PR
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 90
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S43 PR
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 40
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Tet Vector
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 25
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|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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|}
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==<span style="color:darkblue;">blue light receptor</span>==
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===LOV1===
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S33: LOVtap only
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===LOV3===
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S35: is K322999. This sequence is the LOVtap with the tryptophane promoter, RBS, mRFP and two terminators.
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<br/>
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<br/>
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==<span style="color:green;">green light receptor</span>==
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===Gel===
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'''Investigators: Jakob'''
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Comments: Miniprep from June,12 were loaded onto a gel.
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Ccas (M31, M32, M30)in pSB1C3
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[[File:Gimp_Testverdau_12_07_2011_M30-M38.jpg|none|border|caption]]
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Image of the gel of M30-MM32 and M36-M38
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Comments: All the samples were negative.
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<br/>
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<br/>
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==<span style="color:red;">red light receptor</span>==
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===Gel===
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'''Investigators: Jakob'''
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Miniprep from June,12 were loaded onto a gel. If they are positive: Sequencing
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ho1+terminator (M36, M37, M38) in pSB1C3
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Comments: All the samples were negative. See image above.
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==<span style="color:grey;">Precipitator</span>==
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===Plastic binding site===
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'''Investigators: Sophie'''
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Picking colonies (M2a, M2b, M3), inoculating LB (Cm) and let grow overnight. Start: 17:15
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Latest revision as of 00:55, 22 September 2011


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