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Team:Freiburg/Notebook
From 2012.igem.org
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• Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT --> protocol: sanjana et al. (first hexamers...) | • 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 | • transformation: GGC-reaction | ||
| + | |||
• making aliquots of ordered GGC-Primers (freiGEM-method) | • making aliquots of ordered GGC-Primers (freiGEM-method) | ||
| + | |||
• making aliquots of ordered, i.e. synthesized, direpeats | • making aliquots of ordered, i.e. synthesized, direpeats | ||
| - | • extension-PCR of direpeats | + | |
| + | • extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats | ||
| + | |||
• PCR-Purification of extension-PCR | • 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 | • 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 == | == week 06/11/12 - 06/16/12 == | ||
| - | • optimizing PCR conditions for extension of direpeats | + | • optimizing PCR conditions for extension of direpeats |
| - | • redoing GGC-reaction --> protocol: sanjana et al. | + | |
| - | • transformation of redone GGC-reaction | + | • redoing GGC-reaction --> protocol: sanjana et al. |
| - | • GGC á la freiGEM --> transformation | + | |
| - | • testing of iGEM-distribution kit | + | • transformation of redone GGC-reaction |
| + | |||
| + | • GGC á la freiGEM --> transformation | ||
| + | |||
| + | • testing of iGEM-distribution kit | ||
| + | |||
== week 06/17/12 - 06/23/12 == | == week 06/17/12 - 06/23/12 == | ||
| - | • cloning of direpeats into pJET 1.2 vector-system | + | • cloning of direpeats into pJET 1.2 vector-system |
| - | • colony-PCR of freiGEM-GGC product | + | |
| - | • place sequencing order for freiGEM-GGC | + | • colony-PCR of freiGEM-GGC product |
| + | |||
| + | • place sequencing order for freiGEM-GGC | ||
| + | |||
== week 06/24/12 - 07/01/12 == | == week 06/24/12 - 07/01/12 == | ||
| - | • optimizing of freiGEM-GGC under various conditions | + | • 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 | + | • transformation of pJET-direpeats into bacteria |
| - | • Miniprep of GGC-transformation (1 successful) | + | |
| + | • 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 == | == week 07/02/12 - 07/08/12 == | ||
| - | • extension-PCR with all 96 direpeats on one well-plate | + | • extension-PCR with all 96 direpeats on one well-plate |
| - | • transformation | + | |
| - | • PCR-amplification of all 4 iGEM-backbones --> testing different conditions | + | • transformation |
| - | • making bacteria competent for transformation | + | |
| + | • PCR-amplification of all 4 iGEM-backbones --> testing different conditions | ||
| + | |||
| + | • making bacteria competent for transformation | ||
| + | |||
== week 07/09/12 - 07/15/12 == | == week 07/09/12 - 07/15/12 == | ||
| - | • digest of exDirepeats with XbaI and PstI | + | • digest of exDirepeats with XbaI and PstI |
| - | • nanodrop of exDirepeats | + | |
| - | • ligation of exDirepeats in psB1C3 vector backbone and then transformation | + | • nanodrop of exDirepeats |
| - | • colony-PCR, gel run, making cultures | + | |
| - | • miniprep of some of the 96 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 == | == 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 | + | • 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) | + | • amplification of psb1c3 vector backbone, then gel-run and gel-purification |
| - | • miniprep of synthesis-products | + | |
| - | • repeat of pcr-amplification of psb1c3 vector backbone | + | • 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 23/07/12 - 07/29/12 == | ||
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== week 07/30/12 - 08/05/12 == | == 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 | + | • 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 | + | • 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 == | == week 08/06/12 - 08/12/12 == | ||
| - | • 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 | + | |
| - | • gel-run of mutagenesis-PCR of psb1c3 | + | • repeat of GGC to get MammoBrick |
| + | |||
| + | • gel-run of mutagenesis-PCR of psb1c3 | ||
| + | |||
== week 08/13/12 - 08/19/12 == | == week 08/13/12 - 08/19/12 == | ||
Revision as of 11:03, 16 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 in order to produce diefferent restriction sites for correct location of direpeats
• 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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