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Team:Freiburg/Notebook
From 2012.igem.org
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<!--- The Mission, Experiments ---> | <!--- The Mission, Experiments ---> | ||
=<span style="color:#2244AA"> Notebook = | =<span style="color:#2244AA"> Notebook = | ||
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== week 06/04/12 - 06/10/12 == | == week 06/04/12 - 06/10/12 == | ||
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• GGC á la freiGEM --> transformation | • GGC á la freiGEM --> transformation | ||
• testing of iGEM-distribution kit | • testing of iGEM-distribution kit | ||
| - | week 06/17/12 - 06/23/12 | + | |
| + | == week 06/17/12 - 06/23/12 == | ||
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• cloning of direpeats into pJET 1.2 vector-system | • cloning of direpeats into pJET 1.2 vector-system | ||
• colony-PCR of freiGEM-GGC product | • colony-PCR of freiGEM-GGC product | ||
• place sequencing order for freiGEM-GGC | • place sequencing order for freiGEM-GGC | ||
| - | week 06/24/12 - 07/01/12 | + | |
| + | == week 06/24/12 - 07/01/12 == | ||
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• optimizing of freiGEM-GGC under various conditions | • optimizing of freiGEM-GGC under various conditions | ||
• transformation of pJET-direpeats into bacteria | • transformation of pJET-direpeats into bacteria | ||
• using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing | • using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing | ||
• Miniprep of GGC-transformation (1 successful) | • Miniprep of GGC-transformation (1 successful) | ||
| - | week 07/02/12 - 07/08/12 | + | |
| + | == week 07/02/12 - 07/08/12 == | ||
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• extension-PCR with all 96 direpeats on one well-plate | • extension-PCR with all 96 direpeats on one well-plate | ||
• transformation | • transformation | ||
• PCR-amplification of all 4 iGEM-backbones --> testing different conditions | • PCR-amplification of all 4 iGEM-backbones --> testing different conditions | ||
• making bacteria competent for transformation | • making bacteria competent for transformation | ||
| - | week 07/09/12 - 07/15/12 | + | |
| + | == week 07/09/12 - 07/15/12 == | ||
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• digest of exDirepeats with XbaI and PstI | • digest of exDirepeats with XbaI and PstI | ||
• nanodrop of exDirepeats | • nanodrop of exDirepeats | ||
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• colony-PCR, gel run, making cultures | • colony-PCR, gel run, making cultures | ||
• miniprep of some of the 96 exDirepeats | • miniprep of some of the 96 exDirepeats | ||
| - | week 07/16/12 - 07/22/12 | + | |
| + | == week 07/16/12 - 07/22/12 == | ||
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• Transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells | • Transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells | ||
• amplification of psb1c3 vector backbone, then gel-run and gel-purification | • amplification of psb1c3 vector backbone, then gel-run and gel-purification | ||
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• miniprep of synthesis-products | • miniprep of synthesis-products | ||
• repeat of pcr-amplification of psb1c3 vector backbone | • repeat of pcr-amplification of psb1c3 vector backbone | ||
| - | week 23/07/12 - 07/29/12 | + | |
| - | week 07/30/12 - 08/05/12 | + | == week 23/07/12 - 07/29/12 == |
| + | |||
| + | |||
| + | == week 07/30/12 - 08/05/12 == | ||
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• GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone | • GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone | ||
• to do so a extension-PCR on the parts was done | • to do so a extension-PCR on the parts was done | ||
• CMV-Promotor was taken out of iGEM Distribution Kit 2012 | • CMV-Promotor was taken out of iGEM Distribution Kit 2012 | ||
| - | week 08/06/12 - 08/12/12 | + | |
| + | == week 08/06/12 - 08/12/12 == | ||
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• mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI | • mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI | ||
• repeat of GGC to get MammoBrick | • repeat of GGC to get MammoBrick | ||
• gel-run of mutagenesis-PCR of psb1c3 | • gel-run of mutagenesis-PCR of psb1c3 | ||
| - | week 08/13/12 - 08/19/12 | + | |
| + | == week 08/13/12 - 08/19/12 == | ||
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Revision as of 22:07, 15 September 2012
Notebook
week 06/04/12 - 06/10/12
• Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT --> protocol: sanjana et al. (first hexamers...) • transformation: GGC-reaction • making aliquots of ordered GGC-Primers (freiGEM-method) • making aliquots of ordered, i.e. synthesized, direpeats • extension-PCR of direpeats with ordered freiGEM-GGC- • PCR-Purification of extension-PCR • mutagenesis-PCR of Zhang-plasmid to remove restriction sites (SpeI, PstI, EcoRI, XbaI) --> making the plasmid work with iGEM-standard
week 06/11/12 - 06/16/12
• optimizing PCR conditions for extension of direpeats • redoing GGC-reaction --> protocol: sanjana et al. • transformation of redone GGC-reaction • GGC á la freiGEM --> transformation • testing of iGEM-distribution kit
week 06/17/12 - 06/23/12
• cloning of direpeats into pJET 1.2 vector-system • colony-PCR of freiGEM-GGC product • place sequencing order for freiGEM-GGC
week 06/24/12 - 07/01/12
• optimizing of freiGEM-GGC under various conditions • transformation of pJET-direpeats into bacteria • using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing • Miniprep of GGC-transformation (1 successful)
week 07/02/12 - 07/08/12
• extension-PCR with all 96 direpeats on one well-plate • transformation • PCR-amplification of all 4 iGEM-backbones --> testing different conditions • making bacteria competent for transformation
week 07/09/12 - 07/15/12
• digest of exDirepeats with XbaI and PstI • nanodrop of exDirepeats • ligation of exDirepeats in psB1C3 vector backbone and then transformation • colony-PCR, gel run, making cultures • miniprep of some of the 96 exDirepeats
week 07/16/12 - 07/22/12
• Transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells • amplification of psb1c3 vector backbone, then gel-run and gel-purification • picking of colonies (transformation of synthesis-products) • miniprep of synthesis-products • repeat of pcr-amplification of psb1c3 vector backbone
week 23/07/12 - 07/29/12
week 07/30/12 - 08/05/12
• GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone • to do so a extension-PCR on the parts was done • CMV-Promotor was taken out of iGEM Distribution Kit 2012
week 08/06/12 - 08/12/12
• mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI • repeat of GGC to get MammoBrick • gel-run of mutagenesis-PCR of psb1c3
week 08/13/12 - 08/19/12
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