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Team:Freiburg/Notebook
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<!--- The Mission, Experiments ---> | <!--- The Mission, Experiments ---> | ||
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== week 06/04/12 - 06/10/12 == | == week 06/04/12 - 06/10/12 == | ||
Revision as of 22:31, 25 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 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
• repeating the assembly of MammoBrick vector containing CMV Promotor, PostORF-Part and Puromycin-ORF
• repeating mutagenesis-PCR on vector backbone psb1c3
• extPCR of TAL-ORF (synthesis-product) in order to produce a n-terminal kind of adapter to paste recombinase
• producing an provisory mammalian expression vector --> cloning of TAL-ORF into mammalian vector backbone
• to do so the vector and the insert have been digested and then ligated and transformed into dh10b e. coli strain bacteria
• transformation of mutated vector backbone
• colony pcr to test provisory mammalian expression vector
week 08/20/12 - 08/26/12
• repeating mutagenesis pcr for vector backbone psb1c3 with another template
• finding a new method for successful extension pcr and then digest and ligation into the psb1cr backbone
week 08/27/12 - 09/02/12
• finding out that CMV promotor taken out of the registry distribution kit is crap
• trying to get another vector with cmv promotor and ordering new primer pair to amplify it
• after finding a working method for direpeat extension, digestion and ligation into psb1c3 vector backbone we now started to execute it on our synthesized direpeats
week 09/03/12 - 09/09/12
• GGC-reaction in order to produce recombinase-linker-n-tal-orf construct
• annealing of linker-part (was ordered as two single-strand primers)
• after ggc-reaction was done a pcr-amplification on the new assembled part was executed
• gel-run shows a very weak band at 2,7 kb for hyperactive gin recombinase
• GGC-reaction in order to clone direpeats for a complete TAL-Domain into provisory mammalian expression vector
• repeating the extextPCR on the recombinase-linker-n-tal-orf construct under different conditions after the first pcr reaction failed
• amplification of mutated psb1c3 vector backbone
• trying to clone Transcription Factor into provisory mammalian expression vector
• repeating of GGC reaction in order to produce MammoBrick
• still working on our toolkit, doing the final steps: sent them off for sequencing
week 09/10/12 - 09/16/12
• doing the final steps in producing our toolkit: miniprep was done for the last three batches of direpeats, which can finally be sequenced on monday
• we now have 75 successfully sequenced direpeats in the psb1c3 vector backbone, ready to send them off to igem headquarters
• new working cmv promotor is now in psb1c3 vector backbone, too. we now have an improved, i.e. finally working, biobrick for the registry
