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Team:Freiburg/Notebook
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== 04/06 - 10/06 == | == 04/06 - 10/06 == | ||
Revision as of 21:58, 26 September 2012
Notebook
04/06 - 10/06
- Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT
- 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
11/06 - 16/06
- optimizing PCR conditions for extension of direpeats
- redoing GGC-reaction
- transformation of redone GGC-reaction
- GGC á la freiGEM --> transformation
- testing of iGEM-distribution kit
17/06 - 23/06
- cloning of direpeats into pJET 1.2 vector-system
- colony-PCR of freiGEM-GGC product
- place sequencing order for freiGEM-GGC
24/06 - 01/07
- 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)
02/07 - 08/07
- 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
09/07 - 15/07
- 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
16/07 - 22/07
- 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
30/07 - 05/08
- 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
06/08 - 12/08
- 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
13/08 - 19/08
- 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
20/08 - 26/08
- 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
27/08 - 02/09
- finding out that CMV promotor taken out of the registry distribution kit is not good at all
- 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
03/09 - 09/09
- 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
10/09 - 16/09
- 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
