Team:Freiburg/Notebook

From 2012.igem.org

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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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Revision as of 22:03, 15 September 2012




Notebook

September
MTWTFSS
          [http://2012.igem.org/wiki/index.php?title=Freiburg/1_September_2012&action=edit 1] [http://2012.igem.org/wiki/index.php?title=Freiburg/2_September_2012&action=edit 2]
[http://2012.igem.org/wiki/index.php?title=Freiburg/3_September_2012&action=edit 3] [http://2012.igem.org/wiki/index.php?title=Freiburg/4_September_2012&action=edit 4] [http://2012.igem.org/wiki/index.php?title=Freiburg/5_September_2012&action=edit 5] [http://2012.igem.org/wiki/index.php?title=Freiburg/6_September_2012&action=edit 6] [http://2012.igem.org/wiki/index.php?title=Freiburg/7_September_2012&action=edit 7] [http://2012.igem.org/wiki/index.php?title=Freiburg/8_September_2012&action=edit 8] [http://2012.igem.org/wiki/index.php?title=Freiburg/9_September_2012&action=edit 9]
[http://2012.igem.org/wiki/index.php?title=Freiburg/10_September_2012&action=edit 10] [http://2012.igem.org/wiki/index.php?title=Freiburg/11_September_2012&action=edit 11] [http://2012.igem.org/wiki/index.php?title=Freiburg/12_September_2012&action=edit 12] [http://2012.igem.org/wiki/index.php?title=Freiburg/13_September_2012&action=edit 13] [http://2012.igem.org/wiki/index.php?title=Freiburg/14_September_2012&action=edit 14] [http://2012.igem.org/wiki/index.php?title=Freiburg/15_September_2012&action=edit 15] [http://2012.igem.org/wiki/index.php?title=Freiburg/16_September_2012&action=edit 16]
[http://2012.igem.org/wiki/index.php?title=Freiburg/17_September_2012&action=edit 17] [http://2012.igem.org/wiki/index.php?title=Freiburg/18_September_2012&action=edit 18] [http://2012.igem.org/wiki/index.php?title=Freiburg/19_September_2012&action=edit 19] [http://2012.igem.org/wiki/index.php?title=Freiburg/20_September_2012&action=edit 20] [http://2012.igem.org/wiki/index.php?title=Freiburg/21_September_2012&action=edit 21] [http://2012.igem.org/wiki/index.php?title=Freiburg/22_September_2012&action=edit 22] [http://2012.igem.org/wiki/index.php?title=Freiburg/23_September_2012&action=edit 23]
[http://2012.igem.org/wiki/index.php?title=Freiburg/24_September_2012&action=edit 24] [http://2012.igem.org/wiki/index.php?title=Freiburg/25_September_2012&action=edit 25] [http://2012.igem.org/wiki/index.php?title=Freiburg/26_September_2012&action=edit 26] [http://2012.igem.org/wiki/index.php?title=Freiburg/27_September_2012&action=edit 27] [http://2012.igem.org/wiki/index.php?title=Freiburg/28_September_2012&action=edit 28] [http://2012.igem.org/wiki/index.php?title=Freiburg/29_September_2012&action=edit 29] [http://2012.igem.org/wiki/index.php?title=Freiburg/30_September_2012&action=edit 30] 
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


no frame
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