"
Team:Freiburg/Notebook
From 2012.igem.org
| Line 6: | Line 6: | ||
== week 06/04/12 - 06/10/12 == | == week 06/04/12 - 06/10/12 == | ||
| - | • Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT | + | • Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT |
| - | • transformation: GGC-reaction | + | -> protocol: sanjana et al. (first hexamers...) |
| - | • making aliquots of ordered GGC-Primers (freiGEM-method) | + | |
| - | • making aliquots of ordered, i.e. synthesized, direpeats | + | • transformation: GGC-reaction |
| - | • extension-PCR of direpeats with ordered freiGEM-GGC- | + | |
| - | • PCR-Purification of extension-PCR | + | • making aliquots of ordered GGC-Primers (freiGEM-method) |
| - | • mutagenesis-PCR of Zhang-plasmid to remove restriction sites (SpeI, PstI, EcoRI, XbaI) --> making the plasmid work with iGEM-standard | + | • 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 == | == week 06/11/12 - 06/16/12 == | ||
Revision as of 22:10, 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
|
